Methods and systems for sample processing or analysis

ABSTRACT

The present disclosure provides methods and systems for detecting nucleic acid sequences in a biological sample having a three-dimensional matrix. The present disclosure also provides methods and systems for processing a biological sample for use in nucleic acid sequence detection.

CROSS-REFERENCE

This application is a continuation of International Application No.PCT/US19/43773, filed Jul. 26, 2019, which claims priority to U.S.Provisional Patent Application No. 62/711,994, filed Jul. 30, 2018,which is entirely incorporated herein by reference.

BACKGROUND

A padlock probe may be a linear circularizable oligonucleotide which hasfree 5′ and 3′ ends which are available for ligation, to result in theadoption of a circular conformation. For circularization (e.g., byligation) to occur, the padlock probe may have a free 5′ phosphate groupor 5′ adenylated end. To allow the juxtaposition of the ends of thepadlock probe for ligation, the padlock probe may be configured to haveits 5′ and 3′ terminal regions complementary to its target sequence(e.g., a ribonucleic acid or synthesized complementary deoxyribonucleicacid (cDNA) molecule in the cell sample to be analyzed). These regionsof complementarity may allow specific binding of the padlock probe toits target sequence by virtue of hybridization to specific sequences inthe target.

SUMMARY

The present disclosure provides methods and systems for nucleic acidsequence detection in a biological sample having a three-dimensionalmatrix. The present disclosure also provides methods and systems forsample processing for use in target analysis or detection in adownstream application, such as in situ sequencing.

In an aspect, the disclosure provides a method for identification of anucleic acid sequence in a biological sample. In one embodiment, themethod comprises: (a) providing the biological sample comprising aribonucleic acid (RNA) molecule hybridized to a deoxyribonucleic acidmolecule (DNA) in a three-dimensional (3D) matrix, wherein the RNAmolecule comprises the nucleic acid sequence; (b) using a reversetranscriptase to degrade or digest at least a portion of the RNAmolecule hybridized to the DNA molecule, which DNA molecule comprises anadditional nucleic acid sequence that is a reverse complement of thenucleic acid sequence; and (c) detecting the additional nucleic acidsequence in the biological sample, thereby identifying the nucleic acidsequence.

In some embodiments, the DNA molecule is a complementarydeoxyribonucleic acid (cDNA) molecule. In some embodiments, the methodfurther comprises, prior to (a), using an additional reversetranscriptase to reverse transcribe the RNA molecule to generate the DNAmolecule hybridized to the RNA molecule in the biological sample. Insome embodiments, the method further comprises, prior to (a), using thereverse transcriptase to reverse transcribe the RNA molecule to generatethe DNA molecule hybridized to the RNA molecule in the biologicalsample.

In some embodiments, the DNA molecule is immobilized to the 3D matrix.In some embodiments, the DNA molecule comprises a functional moiety, andwherein the DNA molecule is immobilized to the 3D matrix via thefunctional moiety. In some embodiments, the RNA molecule is immobilizedto the 3D matrix. In some embodiments, the RNA molecule comprises afunctional moiety, and wherein the RNA molecule is immobilized to the 3Dmatrix via the functional moiety. In some embodiments, the methodfurther comprises using a matrix-forming material to form the 3D matrix.

In some embodiments, (c) comprises contacting the cDNA molecule with aprobe. In some embodiments, the probe comprises a functional moiety,wherein the probe is immobilized to the 3D matrix via the functionalmoiety. In some embodiments, the probe is a padlock probe, wherein thepadlock probe comprises 5′ and 3′ terminal regions complementary to thecDNA molecule. In some embodiments, the method further compriseshybridizing the 5′ and 3′ terminal regions of the padlock probe to thecDNA molecule. In some embodiments, the method further comprisescircularizing the padlock probe by ligating two ends of the padlockprobe together, to yield a circularized padlock probe. In someembodiments, the two ends of the padlock probe are contiguous. In someembodiments, the two ends of the padlock probe are separated by a gapregion comprising at least one nucleotide. In some embodiments, the gapregion comprises from 2 to 500 nucleotides. In some embodiments, themethod further comprises filling the gap region by incorporating atleast one nucleotide in an extension reaction. In some embodiments, themethod further comprises filling the gap region by at least oneadditional nucleotide or an additional oligonucleotide sequence. In someembodiments, the additional oligonucleotide sequence is from 2 to 500nucleotides in length.

In some embodiments, the method further comprises subjecting thecircularized padlock probe to rolling circle amplification (RCA) togenerate an amplification product of a sequence of the circularizedpadlock probe, which amplification product comprises a nucleic acidsequence corresponding to the nucleic acid sequence of the RNA molecule.In some embodiments, the method further comprises detecting the nucleicacid sequence of the amplification product, thereby identifying thenucleic acid sequence of the RNA molecule.

In some embodiments, the reverse transcriptase or the additional reversetranscriptase has RNA catalytic cleavage activity. In some embodiments,the reverse transcriptase or the additional reverse transcriptase hasRNA catalytic cleavage activity of an RNA/DNA duplex. In someembodiments, the reverse transcriptase or the additional reversetranscriptase is an Avian myeloblastosis virus (AMV) reversetranscriptase, a wild type human immunodeficiency virus-1 (HIV-1)reverse transcriptase, or a Moloney Murine Leukemia Virus (M-MLV)reverse transcriptase.

In some embodiments, the method further comprises, prior to (a),hybridizing a reverse transcription primer to the RNA molecule. In someembodiments, the reverse transcription primer is hybridizable to the 5′terminal region of the padlock probe. In some embodiments, the reversetranscription primer comprises a functional moiety, wherein the cDNAmolecule is immobilized to the 3D matrix via the function moiety. Insome embodiments, the biological sample comprises a plurality of RNAmolecules, which plurality of RNA molecules has a relative 3D spatialrelationship.

In some embodiments, (b) is performed under a first set of conditionsand using an additional reverse transcriptase or the reversetranscriptase to reverse transcribe the RNA molecule is performed undera second set of conditions, wherein the first set of conditions isdifferent than the second set of conditions. In some embodiments, thefirst set of conditions or the second set of conditions is selected fromthe group consisting of pH, temperature, cofactor concentration, andcation concentration. In some embodiments, the cofactor or cationcomprises Mg²⁺, Mn²⁺, Na⁺, ATP, NADPH. In some embodiments, the secondset of conditions inhibits RNase activity of the reverse transcriptase.In some embodiments, the second set of conditions comprises an RNaseinhibitor. In some embodiments, the RNase inhibitor is a small moleculeinhibitor or a polypeptide.

In another aspect, the disclosure provides a method for identificationof a nucleic acid sequence in a biological sample. In one embodiment,the method comprises: (a) providing the biological sample comprising aribonucleic acid (RNA) molecule hybridized to a deoxyribonucleic acidmolecule (DNA) in a three-dimensional (3D) matrix, wherein the RNAmolecule comprises the nucleic acid sequence; (b) using adeoxyribonucleic acid (DNA) binding protein that is not a reversetranscriptase or a ribonuclease to degrade or digest at least a portionof the RNA molecule hybridized to the DNA molecule, which DNA moleculecomprises an additional nucleic acid sequence that is a reversecomplement of the nucleic acid sequence; and (c) detecting theadditional nucleic acid sequence in the biological sample, therebyidentifying the nucleic acid sequence.

In some embodiments, the DNA molecule is a complementarydeoxyribonucleic acid (cDNA) molecule. In some embodiments, the methodfurther comprises, prior to (a), using a reverse transcriptase toreverse transcribe the RNA molecule to generate the DNA moleculehybridized to the RNA molecule in the biological sample.

In some embodiments, the DNA molecule is immobilized to the 3D matrix.In some embodiments, the DNA molecule comprises a functional moiety, andwherein the DNA molecule is immobilized to the 3D matrix via thefunctional moiety. In some embodiments, the RNA molecule is immobilizedto the 3D matrix. In some embodiments, the RNA molecule comprises afunctional moiety, and wherein the RNA molecule is immobilized to the 3Dmatrix via the functional moiety. In some embodiments, the methodfurther comprises using a matrix-forming material to form the 3D matrix.

In some embodiments, (c) comprises contacting the cDNA molecule with aprobe. In some embodiments, the probe comprises a functional moiety,wherein the probe is immobilized to the 3D matrix via the functionalmoiety. In some embodiments, the probe is a padlock probe, wherein thepadlock probe comprises 5′ and 3′ terminal regions complementary to thecDNA molecule, and hybridizing the 5′ and 3′ terminal regions of thepadlock probe to the cDNA molecule. In some embodiments, the methodfurther comprises circularizing the padlock probe by ligating two endsof the padlock probe together, to yield a circularized padlock probe. Insome embodiments, the two ends of the padlock probe are contiguous. Insome embodiments, the two ends of the padlock probe are separated by agap region comprising at least one nucleotide. In some embodiments, thegap region comprises from 2 to 500 nucleotides. In some embodiments, themethod further comprises filling the gap region by incorporating atleast one nucleotide in an extension reaction. In some embodiments, themethod further comprises filling the gap region by at least oneadditional nucleotide or an additional oligonucleotide sequence. In someembodiments, the additional oligonucleotide sequence is from 2 to 500nucleotides in length.

In some embodiments, the method further comprises subjecting thecircularized padlock probe to rolling circle amplification (RCA) togenerate an amplification product of a sequence of the circularizedpadlock probe, which amplification product comprises a nucleic acidsequence corresponding to the nucleic acid sequence of the RNA molecule.In some embodiments, the method further comprises detecting the nucleicacid sequence of the amplification product, thereby identifying thenucleic acid sequence of the RNA molecule.

In some embodiments, the DNA binding protein has RNA catalytic cleavageactivity. In some embodiments, the DNA binding protein stabilizes theDNA molecule. In some embodiments, the DNA binding protein increases amelting temperature of the DNA molecule. In some embodiments, the DNAbinding protein is Sso7d.

In some embodiments, the method further comprises, prior to (a),hybridizing a reverse transcription primer to the RNA molecule. In someembodiments, the reverse transcription primer is hybridizable to the 5′terminal region of the padlock probe. In some embodiments, the reversetranscription primer comprises a functional moiety, wherein the reversetranscription primer or the DNA molecule is immobilized to the 3D matrixvia the function moiety. In some embodiments, the biological samplecomprises a plurality of RNA molecules, which plurality of RNA moleculeshas a relative 3D spatial relationship.

In another aspect, the disclosure provides a method for identificationof a nucleic acid sequence in a biological sample. In one embodiment,the method comprises: (a) providing the biological sample comprising aribonucleic acid (RNA) molecule hybridized to a deoxyribonucleic acidmolecule (DNA) in a three-dimensional (3D) matrix, wherein the RNAmolecule comprises the nucleic acid sequence; (b) non-enzymaticallydegrading at least a portion of the RNA molecule hybridized to the DNAmolecule, which DNA molecule comprises an additional nucleic acidsequence that is a reverse complement of the nucleic acid sequence; (c)contacting the DNA molecule with a probe; and (d) detecting a sequenceof the probe or a derivative thereof, thereby identifying the nucleicacid sequence of the RNA molecule.

In some embodiments, the DNA molecule is a complementarydeoxyribonucleic acid (cDNA) molecule. In some embodiments, the methodfurther comprises, prior to (a), using a reverse transcriptase toreverse transcribe the RNA molecule to generate the DNA moleculehybridized to the RNA molecule in the biological sample. In someembodiments, the DNA molecule is immobilized to the 3D matrix. In someembodiments, the DNA molecule comprises a functional moiety, wherein theDNA molecule is immobilized to the 3D matrix via the functional moiety.In some embodiments, the RNA molecule is immobilized to the 3D matrix.In some embodiments, the RNA molecule comprises a functional moiety,wherein the RNA molecule is immobilized to the 3D matrix via thefunctional moiety.

In some embodiments, the probe is a padlock probe. In some embodiments,the probe comprises a functional moiety, wherein the probe isimmobilized to the 3D matrix via the functional moiety. In someembodiments, the method further comprises using a matrix-formingmaterial to form the 3D matrix. In some embodiments, the padlock probecomprises 5′ and 3′ terminal regions complementary to the DNA molecule.In some embodiments, the method further comprises hybridizing the 5′ and3′ terminal regions of the padlock probe to the DNA molecule.

In some embodiments, the method further comprises circularizing thepadlock probe by coupling two ends of the padlock probe together, toyield a circularized padlock probe, and detecting a nucleic acidsequence of the circularized padlock probe or a derivative thereof,thereby identifying the nucleic acid sequence of the RNA molecule. Insome embodiments, the two ends of the padlock probe are contiguous. Insome embodiments, the two ends of the padlock probe are separated by agap region comprising at least one nucleotide. In some embodiments, thegap region comprises from 2 to 500 nucleotides. In some embodiments, themethod further comprises filling the gap region by incorporating atleast one nucleotide in an extension reaction. In some embodiments, themethod further comprises filling the gap region by at least oneadditional nucleotide or an additional oligonucleotide sequence. In someembodiments, the additional oligonucleotide sequence is from 2 to 500nucleotides in length.

In some embodiments, (c) comprises subjecting the circularized padlockprobe to rolling circle amplification (RCA) to generate an amplificationproduct of a sequence of the circularized padlock probe, whichamplification product comprises a nucleic acid sequence corresponding tothe nucleic acid sequence of the RNA molecule. In some embodiments, (d)comprises detecting the nucleic acid sequence of the amplificationproduct, thereby identifying the nucleic acid sequence of the RNAmolecule.

In some embodiments, the method further comprises, prior to (a),hybridizing a reverse transcription primer to the RNA molecule. In someembodiments, the reverse transcription primer is hybridizable to the 5′terminal region of the padlock probe. In some embodiments, the reversetranscription primer comprises a functional moiety, wherein the DNAmolecule is immobilized to the 3D matrix via the function moiety. Insome embodiments, (b) comprises subjecting the RNA molecule to chemicaldegradation under a condition selected from the group consisting of a pHhaving value from 6 to 14, a temperature from 10° C. to 100° C., in thepresence of a heavy metal ion, in the presence of a divalent cation, andany combination thereof.

In another aspect, the disclosure provides a method for processing abiological sample. In one embodiment, the method comprises: (a)providing the biological sample comprising a ribonucleic acid (RNA)molecule in a three-dimensional (3D) matrix, wherein the RNA moleculecomprises a nucleic acid sequence; (b) hybridizing a primer to the RNAmolecule, which primer does not include a functional moiety forimmobilization to the matrix; (c) using a reverse transcriptase toreverse transcribe the RNA molecule by extending the primer to generatea complementary deoxyribonucleic acid (cDNA) molecule hybridized to theRNA molecule in the biological sample, which cDNA molecule comprises afunctional moiety that immobilizes the cDNA molecule to the 3D matrix.

In some embodiments, the method further comprises degrading the RNAmolecule hybridized to the cDNA molecule, to provide the cDNA moleculeimmobilized to the 3D matrix through the functional moiety, which cDNAmolecule comprises an additional nucleic acid sequence that is a reversecomplement of the nucleic acid sequence. In some embodiments, the RNAmolecule comprises a functional moiety, wherein the RNA molecule isimmobilized to the 3D matrix via the functional moiety.

In some embodiments, degrading comprises degrading the RNA molecule by anon-ribonuclease enzyme. In some embodiments, the non-ribonucleaseenzyme is a reverse transcriptase or a DNA binding protein. In someembodiments, degrading comprises degrading the RNA molecule by anon-enzymatic reaction. In some embodiments, the non-enzymatic reactionis under a condition selected from the group consisting of a pH havingvalue from 6 to 14, a temperature from 10° C. to 100° C., in thepresence of a heavy metal ion, in the presence of a divalent cation, andany combination thereof.

In some embodiments, the method further comprises contacting the cDNAmolecule with a probe. In some embodiments, the probe comprises a regionthat is not hybridizable with the cDNA molecule. In some embodiments,the probe is a padlock probe, wherein the padlock probe comprises 5′ and3′ terminal regions complementary to the cDNA molecule, and hybridizingthe 5′ and 3′ terminal regions of the padlock probe to the cDNAmolecule. In some embodiments, the method further comprisescircularizing the padlock probe by coupling two ends of the padlockprobe together, to yield a circularized padlock probe, and detecting anucleic acid sequence of the circularized padlock probe or a derivativethereof, thereby identifying the nucleic acid sequence of the RNAmolecule. In some embodiments, the two ends of the padlock probe arecontiguous. In some embodiments, the two ends of the padlock probe areseparated by a gap region comprising at least one nucleotide. In someembodiments, the gap region comprises from 2 to 500 nucleotides. In someembodiments, the method further comprises filling the gap region byincorporating at least one nucleotide in an extension reaction. In someembodiments, the method further comprises filling the gap region by atleast one additional nucleotide or an additional oligonucleotidesequence. In some embodiments, the additional oligonucleotide sequenceis from 2 to 500 nucleotides in length. In some embodiments, the methodfurther comprises subjecting the circularized padlock probe to rollingcircle amplification (RCA) to generate an amplification product of asequence of the circularized padlock probe, which amplification productcomprises a nucleic acid sequence corresponding to the nucleic acidsequence of the RNA molecule. In some embodiments, the method furthercomprises detecting the nucleic acid sequence of the amplificationproduct, thereby identifying the nucleic acid sequence of the RNAmolecule. In some embodiments, the probe comprises a functional moiety,wherein the probe is immobilized to the 3D matrix via the functionalmoiety. In some embodiments, the functional moiety is directlyconjugated on the probe. In some embodiments, the probe hybridizes to atethering oligonucleotide comprising the functional moiety. In someembodiments, the tethering oligonucleotide hybridizes to the region ofthe probe that is not hybridizable to the cDNA molecule. In someembodiments, the method further comprises detecting a sequence of theprobe or a derivative thereof, thereby identifying the nucleic acidsequence of the RNA molecule.

In some embodiments, (c) comprises using the reverse transcriptase toincorporate a nucleotide analog comprising the functional moiety into agrowing strand, to yield the cDNA molecule comprising the nucleotide. Insome embodiments, the nucleotide analog comprises amino-allyl dUTP,5-TCO-PEG4-dUTP, C8-Alkyne-dUTP, 5-Azidomethyl-dUTP, 5-Vinyl-dUTP,5-Ethynyl dUTP, or a combination thereof. In some embodiments, themethod further comprises, subsequent to (b), modifying the primer or thecDNA molecule to include the functional moiety. In some embodiments, theprimer is modified to include the functional moiety prior to generatingthe cDNA molecule. In some embodiments, the primer comprises a regionthat is not hybridizable to the RNA molecule, wherein the regionhybridizes to an additional tethering oligonucleotide that comprises thefunctional moiety.

In some embodiments, (c) comprises attaching the functional moiety tothe cDNA molecule through an enzymatic reaction or a non-enzymaticreaction. In some embodiments, the enzymatic reaction comprises using anenzyme to attach a nucleotide or an oligonucleotide having thefunctional moiety to the cDNA molecule. In some embodiments, the enzymeis a ligase, a polymerase, or a combination thereof. In someembodiments, the non-enzymatic reaction comprises attaching a chemicalreagent having the functional moiety to the cDNA molecule by alkylationor oxymercuration. In some embodiments, the cDNA molecule furtherhybridizes to a tethering oligonucleotide having the functional moiety.In some embodiments, the tethering oligonucleotide hybridizes to theprimer. In some embodiments, the 3D matrix further comprises anadditional functional moiety, which additional functional moiety reactswith the function moiety of the cDNA molecule, thereby immobilizing thecDNA molecule.

Another aspect of the present disclosure provides a non-transitorycomputer readable medium comprising machine executable code that, uponexecution by one or more computer processors, implements any of themethods above or elsewhere herein.

Another aspect of the present disclosure provides a system comprisingone or more computer processors and computer memory coupled thereto. Thecomputer memory comprises machine executable code that, upon executionby the one or more computer processors, implements any of the methodsabove or elsewhere herein.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 shows an example of a method for identification of a nucleic acidsequence in a biological sample.

FIG. 2 shows an example of a method for identification of a nucleic acidsequence in a biological sample.

FIG. 3 shows an example of a method for identification of a nucleic acidsequence in a biological sample.

FIG. 4 shows an example of a method for processing a biological sample.

FIG. 5 shows a computer system that is programmed or otherwiseconfigured to implement methods provided herein.

FIG. 6 shows an example image of a tissue sample processed and imagedusing methods of the present disclosure.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

As used in the specification and claims, the singular form “a”, “an” or“the” includes plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

As used herein, the terms “amplifying” and “amplification” generallyrefer to generating one or more copies (or “amplified product” or“amplification product”) of a nucleic acid. The one or more copies maybe generated by nucleic acid extension. Such extension may be a singleround of extension or multiple rounds of extension. The amplifiedproduct may be generated by polymerase chain reaction (PCR).

The term “reverse transcription,” as used herein, generally refers tothe generation of deoxyribonucleic acid (DNA) from a ribonucleic acid(RNA) template via the action of a reverse transcriptase. Reversetranscription PCR (or RT-PCR) refers to reverse transcription coupledwith PCR.

The term “nucleic acid,” as used herein, generally refers to a polymericform of nucleotides of any length. A nucleic acid may comprise eitherdeoxyribonucleotides (dNTPs) or ribonucleotides (rNTPs), or analogsthereof. A nucleic acid may be an oligonucleotide or a polynucleotide.Nucleic acids may have any three-dimensional structure and may performany function. Non-limiting examples of nucleic acids include DNA, RNA,coding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer RNA, ribosomal RNA, short interfering RNA (siRNA),short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA,recombinant nucleic acids, branched nucleic acids, plasmids, vectors,isolated DNA of any sequence, isolated RNA of any sequence, nucleic acidprobes, and primers. A nucleic acid may comprise one or more modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be made before orafter assembly of the nucleic acid. The sequence of nucleotides of anucleic acid may be interrupted by non-nucleotide components. A nucleicacid may be further modified after polymerization, such as byconjugation, with a functional moiety for immobilization.

As used herein, the term “subject,” generally refers to an entity or amedium that has or may have testable or detectable genetic information.A subject can be a person or an individual. A subject can be avertebrate, such as, for example, a mammal. Non-limiting examples ofmammals include murines, simians, and humans. A subject may be ananimal, such as a farm animal. A subject may be a pet, such as dog, cat,mouse, rat, or bird. Other examples of subjects include food, plant,soil, and water. A subject may be displaying or symptomatic with respectto a disease. As an alternative, the subject may be asymptomatic withrespect to the disease.

Any suitable biological sample that comprises nucleic acid may beobtained from a subject. Any suitable biological sample that comprisesnucleic acid may be used in the methods and systems described herein. Abiological sample may be solid matter (e.g., biological tissue) or maybe a fluid (e.g., a biological fluid). In general, a biological fluidcan include any fluid associated with living organisms. Non-limitingexamples of a biological sample include blood (or components ofblood—e.g., white blood cells, red blood cells, platelets) obtained fromany anatomical location (e.g., tissue, circulatory system, bone marrow)of a subject, cells obtained from any anatomical location of a subject,skin, heart, lung, kidney, breath, bone marrow, stool, semen, vaginalfluid, interstitial fluids derived from tumorous tissue, breast,pancreas, cerebral spinal fluid, tissue, throat swab, biopsy, placentalfluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, cavity fluids, sputum, pus,micropiota, meconium, breast milk, prostate, esophagus, thyroid, serum,saliva, urine, gastric and digestive fluid, tears, ocular fluids, sweat,mucus, earwax, oil, glandular secretions, spinal fluid, hair,fingernails, skin cells, plasma, nasal swab or nasopharyngeal wash,spinal fluid, cord blood, emphatic fluids, and/or other excretions orbody tissues. A biological sample may be a cell-free sample. Suchcell-free sample may include DNA and/or RNA.

Overview

Provided herein are methods and systems for sample processing for use intarget analysis or detection. Methods and systems of the presentdisclosure may be used for various applications, such as in situsequencing or sequence identification (e.g., sequencing or sequenceidentification within a sample, such as, for example, a cell). In thesemethods and systems, probes may be used for target capture andsubsequently for analysis or detection in the sample. Such probes may bepadlock probes. Padlock probes can be designed or configured to bindspecifically to targets. In some cases, padlock probes can be designedto hybridize with targets directly. In some other cases, padlock probescan be designed to bind the target indirectly by hybridizing withmolecules derived from the targets. For example, in some applications inwhich ribonucleic acid (RNA) molecules are the targets, complementarydeoxyribonucleic acid (cDNA) molecules can be synthesized from the RNAtargets by reverse transcription, and the padlock probes can be designedto bind to the cDNA molecules. By hybridization to the cDNA molecule,the ends of the padlock probe are brought into juxtaposition forligation. The ligation may be direct or indirect. In other words, theends of the padlock probe may be ligated directly to each other or theymay be ligated to an intervening nucleic acid molecule or a sequence ofnucleotides. Thus, the terminal regions of the padlock probe may becomplementary to adjacent, or contiguous, regions in the cDNA moleculesynthesized from the RNA target molecule, or they may be complementaryto non-adjacent or non-contiguous regions of the cDNA. In the caseswhere the padlock probe is complementary to non-adjacent ornon-contiguous regions of the cDNA molecule, for ligation to occur, the“gap” between the two ends of the hybridized padlock probe can be filledby an intervening oligonucleotide molecule or a sequence of nucleotides.

Upon addition to a sample having a target molecule, the ends of thepadlock probe may hybridize to complementary regions in a targetmolecule or derivative thereof (e.g., cDNA molecule). Followinghybridization, the padlock probe may be circularized by direct orindirect ligation of the ends of the padlock probe by a ligase enzyme.The circularized padlock probe may be subjected to amplification togenerate an amplification product. For example, the circulated padlockprobe may be subjected to rolling circle amplification (RCA) to generatea DNA nanoball (i.e., rolony). The circularized padlock probe may beprimed by the 3′ end of the cDNA (i.e., the RCA is target-primed). A DNApolymerase with 3′-5′ exonuclease activity may be used. This can permitthe digestion of the cDNA strand in a 3′-5′ direction to a pointadjacent to the bound padlock probe. Alternatively, the cDNA may be ofappropriate length and may act as the primer for the DNApolymerase-mediated amplification reaction without such digestion. As afurther alternative, instead of priming the RCA with the cDNA molecule,an additional primer that can hybridize to the padlock probe may beadded in the sample and used for amplification reaction.

The amplification product (e.g., rolony) can be used for the purpose ofin situ (e.g., within a sample, such as, for example, a cell) moleculardetection by fluorescent in situ sequencing (FISSEQ) in a biologicalsample, such as a cell or a tissue. The biological sample may comprise athree-dimensional matrix (3D matrix). The 3D matrix may be formed bysubjecting the biological sample to a fixing agent, such asformaldehyde. The 3D matrix may also be formed by a matrix-formingmaterial, such as polymerizable monomers or cross-linkable polymers. Theamplification product can serve as an amplified sequencing template forFISSEQ, in which, for example, sequence features of the amplificationproduct can be detected in situ by fluorescent sequencing, including butnot limited to sequencing by synthesis (SBS), sequencing by ligation(SBL), or sequencing by hybridization (SBH). Using a plurality ofpadlock probes, a number of target nucleic acids can be detected in amultiplex manner.

Methods or systems utilizing a ligation reaction of a DNA-DNA duplextemplate formed between a cDNA molecule and a DNA padlock probe can begenerally more efficient than methods or systems utilizing a ligationreaction of a DNA-RNA “hybrid” duplex template formed between an RNAmolecule and a DNA padlock probe. This may be due to the enhancedefficiency of enzymatic ligation between DNA-DNA duplex templatescompared to DNA-RNA hybrid duplex templates. Therefore, all or part of atarget RNA molecule may be first converted into a cDNA molecule, such asby reverse transcription, prior to hybridization with the padlock probe.After generating the cDNA molecule, the RNA molecule can be degraded.Methods and systems provided herein use several methods to degrade RNAmolecule. In some aspects, an enzymatic digestion of the RNA using anon-ribonuclease enzyme with ribonuclease activity is provided. In someother aspects, chemical decomposition of the RNA under conditionswherein cDNA remains substantially chemically stable is provided. Insome cases, the target RNA molecule may directly hybridize to a padlockprobe without prior reverse transcription.

Furthermore, in some applications, methods and systems for sampleprocessing may preserve spatial information associated with each targetmolecule. Such spatial information may be preserved in a biologicalsample having a 3D matrix. To preserve the spatial informationassociated with each RNA molecule being detected in a padlock probeassay, the cDNA molecule can be spatially immobilized within thebiological sample at the original position of the RNA molecule. In thepresent disclosure, several methods are provided to immobilize the cDNAmolecule in a three-dimensional matrix.

Target

Provided herein are methods and systems for sample processing for use intarget analysis or detection. The target may be an analyte of interestin a biological sample. In some cases, the target may be a nucleic acidtarget. In some cases, the target may be a protein. In the cases wherethe target is a protein, a binding agent which binds to the protein canbe linked to a nucleic acid sequence which can then be detected by themethods and systems provided herein. For example, the binding agent canbe a nucleic acid barcode conjugated antibody or antibody fragment. Thenucleic acid target can be a ribonucleic acid (RNA) or adeoxyribonucleic acid (DNA). The nucleic acid target may be naturallyoccurring nucleic acids or non-naturally occurring nucleic acids, suchas nucleic acids that have been made using synthetic methods.

The nucleic acid targets, whether naturally occurring or synthetic, canbe present within a three-dimensional (3D) matrix and covalentlyattached to the 3D matrix such that the relative position of eachnucleic acid is fixed (e.g., immobilized) within the 3D matrix. In thismanner, a 3D matrix of covalently bound nucleic acids of any sequencecan be provided. Each nucleic acid may have its own three-dimensionalcoordinates within the matrix material and each nucleic acid mayrepresent information. In this manner, a large amount of information canbe stored in a 3D matrix. Individual information-encoding nucleic acidtarget, such as DNA or RNA can be amplified and sequenced in situ (i.e.,within the matrix), thereby enabling a large amount of information to bestored and read in a suitable 3D matrix. Naturally occurring nucleicacid targets can include endogenous DNAs and RNAs. Synthetic nucleicacid targets can include primers, barcodes, amplification products andprobes. The synthetic nucleic acid targets may be derived from theendogenous nucleic acid molecules or include sequence information of theendogenous nucleic acid molecules. The synthetic nucleic acid targetscan be used to capture endogenous nucleic acid targets to the 3D matrixand can be subsequently sequenced or detected to identity the sequenceinformation and/or positional (or spatial) information of the endogenousnucleic acid molecules. For example, a synthetic nucleic acid target canbe a primer having a poly-deoxythymine (dT) sequence, which canhybridize to an endogenous mRNA molecule. The primer may be immobilizedto the 3D matrix and may be extended to include sequence information(e.g., a sequence) of the mRNA molecule. The extended primer can then becaptured by padlock probes and amplified in situ for detection. Inanother example, a synthetic nucleic acid target can be a barcodeconjugated on an antibody. The barcode may be captured by padlock probesand amplified in situ for detection.

The nucleic acid target can be an endogenous nucleic acid in abiological sample, for example, genomic DNA, messenger RNA (mRNA),ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), smallcytoplasmic RNA (scRNA), and small nuclear RNA (snRNA). The nucleic acidtarget may be a synthetic nucleic acid linked to a binding agent. Thebinding agent may bind to any biological molecules to be detected in abiological sample. For example, to detect a protein, the binding agentmay be an antibody or a portion thereof having a nucleic acid sequencelinked thereto. For another example, to detect a protein, the bindingagent may be an aptamer.

The nucleic acid target may be amplified to produce amplificationproducts or amplicons within the 3D matrix. The nucleic acid target maybe amplified using nucleic acid amplification, such as, for example,polymerase chain reaction (PCR). The nucleic acid target may be bound toa probe and the probe may be subsequently amplified to produceamplification products or amplicons. In some cases, the nucleic acidtarget is a RNA target, and the RNA target may be reverse transcribed togenerate a cDNA. The cDNA may then be subjected to amplification or maybe contacted with a probe (e.g., a padlock probe). The probe canhybridize with the cDNA. In some cases, the nucleic acid target is a DNAtarget, and the DNA target can be subjected to amplification or can becontacted with a probe (e.g., a padlock probe). For example, the DNAtarget can be amplified directly by an amplification primer. For anotherexample, a padlock probe may be contacted with the DNA target andhybridize to the DNA target. The padlock probe can then be circularizedand amplified. The amplification products or amplicons can be attachedto the matrix, for example, by copolymerization or cross-linking. Thiscan result in a structurally stable and chemically stable 3D matrix ofnucleic acids. The 3D matrix of nucleic acids may allow for prolongedinformation storage and read-out cycles. The nucleic acid/ampliconmatrix may allow for high throughput sequencing of a wide-ranging arrayof samples in three dimensions.

Three-Dimensional Matrix

The present disclosure provides a three-dimensional (3D) matrix. The 3Dmatrix may comprise a plurality of nucleic acids. The 3D matrix maycomprise a plurality of nucleic acids covalently or non-covalentlyattached thereto.

In some cases, a matrix-forming material may be used to form the 3Dmatrix. The matrix forming material may be polymerizable monomers orpolymers, or cross-linkable polymers. The matrix forming material may bepolyacrylamide, acrylamide monomers, cellulose, alginate, polyamide,agarose, dextran, or polyethylene glycol. The matrix forming materialscan form a matrix by polymerization and/or crosslinking of the matrixforming materials using methods specific for the matrix formingmaterials and methods, reagents and conditions. The matrix formingmaterial may form a polymeric matrix. The matrix forming material mayform a polyelectrolyte gel. The matrix forming material may form ahydrogel gel matrix.

The matrix-forming material may form a 3D matrix including the pluralityof nucleic acids while maintaining the spatial relationship of thenucleic acids. In this aspect, the plurality of nucleic acids can beimmobilized within the matrix material. The plurality of nucleic acidsmay be immobilized within the matrix material by co-polymerization ofthe nucleic acids with the matrix-forming material. The plurality ofnucleic acids may also be immobilized within the matrix material bycrosslinking of the nucleic acids to the matrix material or otherwisecross-linking with the matrix-forming material. The plurality of nucleicacids may also be immobilized within the matrix by covalent attachmentor through ligand-protein interaction to the matrix.

According to one aspect, the matrix can be porous thereby allowing theintroduction of reagents into the matrix at the site of a nucleic acidfor amplification of the nucleic acid. A porous matrix may be madeaccording to various methods. For example, a polyacrylamide gel matrixcan be co-polymerized with acrydite-modified streptavidin monomers andbiotinylated DNA molecules, using a suitable acrylamide:bis-acrylamideratio to control the cross-linking density. Additional control over themolecular sieve size and density can be achieved by adding additionalcross-linkers such as functionalized polyethylene glycols.

According to one aspect, the 3D matrix may be sufficiently opticallytransparent or may have optical properties suitable for standardsequencing chemistries and deep three-dimensional imaging for highthroughput information readout. Examples of the sequencing chemistriesthat utilize fluorescence imaging include ABI SoLiD (Life Technologies),in which a sequencing primer on a template is ligated to a library offluorescently labeled octamers with a cleavable terminator. Afterligation, the template can then be imaged using four color channels(FITC, Cy3, Texas Red and Cy5). The terminator can then be cleaved offleaving a free-end to engage in the next ligation-extension cycle. Afterall dinucleotide combinations have been determined, the images can bemapped to the color code space to determine the specific base calls pertemplate. The workflow can be achieved using an automated fluidics andimaging device (i.e., SoLiD 5500 W Genome Analyzer, ABI LifeTechnologies). Another example of sequencing platform uses sequencing bysynthesis, in which a pool of single nucleotide with a cleavableterminator can be incorporated using DNA polymerase. After imaging, theterminator can be cleaved and the cycle can be repeated. Thefluorescence images can then be analyzed to call bases for each DNAamplicons within the flow cell (HiSeq, Illumina).

In some aspects, a biological sample may be fixed in the presence of thematrix-forming materials, for example, hydrogel subunits. By “fixing”the biological sample, it is meant exposing the biological sample, e.g.,cells or tissues, to a fixation agent such that the cellular componentsbecome crosslinked to one another. By “hydrogel” or “hydrogel network”is meant a network of polymer chains that are water-insoluble, sometimesfound as a colloidal gel in which water is the dispersion medium. Inother words, hydrogels are a class of polymeric materials that canabsorb large amounts of water without dissolving. Hydrogels can containover 99% water and may comprise natural or synthetic polymers, or acombination thereof. Hydrogels may also possess a degree of flexibilityvery similar to natural tissue, due to their significant water content.By “hydrogel subunits” or “hydrogel precursors” refers to hydrophilicmonomers, prepolymers, or polymers that can be crosslinked, or“polymerized”, to form a 3D hydrogel network. Without being bound by anyscientific theory, fixation of the biological sample in the presence ofhydrogel subunits may crosslink the components of the biological sampleto the hydrogel subunits, thereby securing molecular components inplace, preserving the tissue architecture and cell morphology.

In some cases, the biological sample (e.g., cell) may be permeabilizedor otherwise made accessible to an environment external to thebiological sample. In some cases, the biological sample may be fixed andpermeabilized first, and then a matrix-forming material can then beadded into the biological sample.

Any convenient fixation agent, or “fixative,” may be used to fix thebiological sample in the absence or in the presence of hydrogelsubunits, for example, formaldehyde, paraformaldehyde, glutaraldehyde,acetone, ethanol, methanol, etc. Typically, the fixative may be dilutedin a buffer, e.g., saline, phosphate buffer (PB), phosphate bufferedsaline (PBS), citric acid buffer, potassium phosphate buffer, etc.,usually at a concentration of about 1-10%, e.g. 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, or 10%, for example, 4% paraformaldehyde/0.1M phosphate buffer;2% paraformaldehyde/0.2% picric acid/0.1M phosphate buffer; 4%paraformaldehyde/0.2% periodate/1.2% lysine in 0.1 M phosphate buffer;4% paraformaldehyde/0.05% glutaraldehyde in phosphate buffer; etc. Thetype of fixative used and the duration of exposure to the fixative willdepend on the sensitivity of the molecules of interest in the specimento denaturation by the fixative, and may be readily determined usingconventional histochemical or immunohistochemical techniques.

The fixative/hydrogel composition may comprise any hydrogel subunits,such as, but not limited to, poly(ethylene glycol) and derivativesthereof (e.g. PEG-diacrylate (PEG-DA), PEG-RGD), polyaliphaticpolyurethanes, polyether polyurethanes, polyester polyurethanes,polyethylene copolymers, polyamides, polyvinyl alcohols, polypropyleneglycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide,poly(hydroxyethyl acrylate), and poly(hydroxyethyl methacrylate),collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin,alginate, protein polymers, methylcellulose and the like. Agents such ashydrophilic nanoparticles, e.g., poly-lactic acid (PLA), poly-glycolicacid (PLG), poly(lactic-co-glycolic acid) (PLGA), polystyrene,poly(dimethylsiloxane) (PDMS), etc. may be used to improve thepermeability of the hydrogel while maintaining patternability. Materialssuch as block copolymers of PEG, degradable PEO, poly(lactic acid)(PLA), and other similar materials can be used to add specificproperties to the hydrogel. Crosslinkers (e.g. bis-acrylamide,diazirine, etc.) and initiators (e.g. azobisisobutyronitrile (AIBN),riboflavin, L-arginine, etc.) may be included to promote covalentbonding between interacting macromolecules in later polymerizationsteps.

The biological sample (e.g., a cell or tissue) may be permeabilizedafter being fixed. Permeabilization may be performed to facilitateaccess to cellular cytoplasm or intracellular molecules, components orstructures of a cell. Permeabilization may allow an agent (such as aphospho-selective antibody, a nucleic acid conjugated antibody, anucleic acid probe, a primer, etc.) to enter into a cell and reach aconcentration within the cell that is greater than that which wouldnormally penetrate into the cell in the absence of such permeabilizingtreatment. In some embodiments, cells may be stored followingpermeabilization. In some cases, the cells may be contacted with one ormore agents to allow penetration of the one or more agent afterpermeabilization without any storage step and then analyzed. In someembodiments, cells may be permeabilized in the presence of at leastabout 60%, 70%, 80%, 90% or more methanol (or ethanol) and incubated onice for a period of time. The period of time for incubation can be atleast about 10, 15, 20, 25, 30, 35, 40, 50, 60 or more minutes.

In some embodiments, permeabilization of the cells may be performed byany suitable method. Selection of an appropriate permeabilizing agentand optimization of the incubation conditions and time may be performed.Suitable methods include, but are not limited to, exposure to adetergent (such as CHAPS, cholic acid, deoxycholic acid, digitonin,n-dodecyl-beta-D-maltoside, lauryl sulfate, glycodeoxycholic acid,n-lauroylsarcosine, saponin, and triton X-100) or to an organic alcohol(such as methanol and ethanol). Other permeabilizing methods cancomprise the use of certain peptides or toxins that render membranespermeable. Permeabilization may also be performed by addition of anorganic alcohol to the cells.

Permeabilization can also be achieved, for example, by way ofillustration and not limitation, through the use of surfactants,detergents, phospholipids, phospholipid binding proteins, enzymes, viralmembrane fusion proteins and the like; through the use of osmoticallyactive agents; by using chemical crosslinking agents; by physicochemicalmethods including electroporation and the like, or by otherpermeabilizing methodologies.

Thus, for instance, cells may be permeabilized using any of a variety ofknown techniques, such as exposure to one or more detergents (e.g.,digitonin, Triton X-100™, NP-40™, octyl glucoside and the like) atconcentrations below those used to lyse cells and solubilize membranes(i.e., below the critical micelle concentration). Certain transfectionreagents, such as dioleoyl-3-trimethylammonium propane (DOTAP), may alsobe used. ATP can also be used to permeabilize intact cells. Lowconcentrations of chemicals used as fixatives (e.g., formaldehyde) mayalso be used to permeabilize intact cells.

The nucleic acids (e.g., RNA molecule, cDNA molecule, primer, or probe)described herein may comprise a functional moiety. The nucleic acids canbe linked to the 3D matrix by the functional moiety. The functionalmoiety can be reacted with a reactive group on the 3D matrix throughconjugation chemistry. In some cases, the functional moiety can beattached to target of interest through conjugation chemistry. In somecases, the functional moiety can be directly attached to a reactivegroup on the native nucleic acid molecule. In some cases, the functionalmoiety can be indirectly linked to a target through an intermediatechemical or group. The conjugation strategies described herein are notlimited to nucleic acid targets and can be used for protein or smallmolecule targets as well. A nucleotide analog comprising a functionalmoiety may be incorporated into a growing chain of the nucleic acid(e.g., cDNA molecule, probe, or primer) during nucleic acid synthesis oran extension reaction.

As used herein, the term “reactive group” or “functional moiety” meansany moiety on a first reactant that is capable of reacting chemicallywith another functional moiety or reactive group on a second reactant toform a covalent or ionic linkage. “Reactive group” and “functionalmoiety” may be used interchangeably. For example, a reactive group ofthe monomer or polymer of the matrix-forming material can reactchemically with a functional moiety (or another reactive group) on thesubstrate of interest or the target to form a covalent or ionic linkage.The substrate of interest or the target may then be immobilized to thematrix via the linkage formed by the reactive group and the functionalmoiety. Examples of suitable reactive groups or functional moietiesinclude electrophiles or nucleophiles that can form a covalent linkageby reaction with a corresponding nucleophile or electrophile,respectively, on the substrate of interest. Non-limiting examples ofsuitable electrophilic reactive groups may include, for example, estersincluding activated esters (such as, for example, succinimidyl esters),amides, acrylamides, acyl azides, acyl halides, acyl nitriles,aldehydes, ketones, alkyl halides, alkyl sulfonates, anhydrides, arylhalides, aziridines, boronates, carbodiimides, diazoalkanes, epoxides,haloacetamides, haloplatinates, halotriazines, imido esters,isocyanates, isothiocyanates, maleimides, phosphoramidites, silylhalides, sulfonate esters, sulfonyl halides, and the like. Non-limitingexamples of suitable nucleophilic reactive groups may include, forexample, amines, anilines, thiols, alcohols, phenols, hyrazines,hydroxylamines, carboxylic acids, glycols, heterocycles, and the like.

The present disclosure provides a method of modifying a nucleic acid insitu to comprise a functional moiety. In some cases, the functionalmoiety may comprise a polymerizeable group. In some cases, thefunctional moiety may comprise a free radical polymerizeable group. Insome cases, the functional moiety may comprise an amine, a thiol, anazide, an alkyne, a nitrone, an alkene, a tetrazine, a tetrazole, anacrydite or other click reactive group. In some cases, the functionalmoiety can be subsequently linked to a 3D matrix in situ. The functionalmoiety may further be used to preserve the absolute or relative spatialrelationships among two or more molecules within a sample.

The biological sample within the 3D matrix may be cleared of proteinsand/or lipids that are not targets of interest. For example, thebiological sample can be cleared of proteins (also called“deproteination”) by enzymatic proteolysis. The clearing step may beperformed before or after covalent immobilization of any targetmolecules or derivatives thereof.

In some cases, the clearing step is performed after covalentimmobilization of target nucleic acid molecules (e.g., RNA or DNA),primers (e.g., RT primers), derivatives of target molecules (e.g., cDNAor amplicons), probes (e.g., padlock probes) to a synthetic 3D matrix.Performing the clearing step after immobilization can enable anysubsequent nucleic acid hybridization reactions to be performed underconditions where the sample has been substantially deproteinated, as byenzymatic proteolysis (“protein clearing”). This method can have thebenefit of removing ribosomes and other RNA- ornucleic-acid-target-binding proteins from the target molecule (whilemaintaining spatial location), where the protein component may impede orinhibit primer binding, reverse transcription, or padlock ligation andamplification, thereby improving the sensitivity and quantitativity ofthe assay by reducing bias in probe capture events due to proteinoccupation of or protein crowding/proximity to the target nucleic acid.

The clearing step can comprise removing non-targets from the 3D matrix.The clearing step can comprise degrading the non-targets. The clearingstep can comprise exposing the sample to an enzyme (e.g., a protease)able to degrade a protein. The clearing step can comprise exposing thesample to a detergent.

Proteins may be cleared from the sample using enzymes, denaturants,chelating agents, chemical agents, and the like, which may break downthe proteins into smaller components and/or amino acids. These smallercomponents may be easier to remove physically, and/or may besufficiently small or inert such that they do not significantly affectthe background. Similarly, lipids may be cleared from the sample usingsurfactants or the like. In some cases, one or more of these agents areused, e.g., simultaneously or sequentially. Non-limiting examples ofsuitable enzymes include proteinases such as proteinase K, proteases orpeptidases, or digestive enzymes such as trypsin, pepsin, orchymotrypsin. Non-limiting examples of suitable denaturants includeguanidine HCl, acetone, acetic acid, urea, or lithium perchlorate.Non-limiting examples of chemical agents able to denature proteinsinclude solvents such as phenol, chloroform, guanidinium isocyananate,urea, formamide, etc. Non-limiting examples of surfactants includeTriton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenylether), SDS (sodium dodecyl sulfate), Igepal CA-630, or poloxamers.Non-limiting examples of chelating agents includeethylenediaminetetraacetic acid (EDTA), citrate, or polyaspartic acid.In some embodiments, compounds such as these may be applied to thesample to clear proteins, lipids, and/or other components. For instance,a buffer solution (e.g., containing Tris ortris(hydroxymethyl)aminomethane) may be applied to the sample, thenremoved.

In some cases, nucleic acids that are not target of interest may also becleared. These non-target nucleic acids may not be captured and/orimmobilized to the 3D matrix, and therefore can be removed with anenzyme to degrade nucleic acid molecules. Non-limiting examples of DNAenzymes that may be used to remove DNA include DNase I, dsDNase, avariety of restriction enzymes, etc. Non-limiting examples of techniquesto clear RNA include RNA enzymes such as RNase A, RNase T, or RNase H,or chemical agents, e.g., via alkaline hydrolysis (for example, byincreasing the pH to greater than 10). Non-limiting examples of systemsto remove sugars or extracellular matrix include enzymes such aschitinase, heparinases, or other glycosylases. Non-limiting examples ofsystems to remove lipids include enzymes such as lipidases, chemicalagents such as alcohols (e.g., methanol or ethanol), or detergents suchas Triton X-100 or sodium dodecyl sulfate. In this way, the backgroundof the sample may be removed, which may facilitate analysis of thenucleic acid probes or other targets, e.g., using fluorescencemicroscopy, or other techniques as described herein.

Solid Support

A matrix may be used in conjunction with a solid support. For example,the matrix can be polymerized in such a way that one surface of thematrix is attached to a solid support (e.g., a glass surface, a flowcell, a glass slide, a well), while the other surface of the matrix isexposed or sandwiched between two solid supports. According to oneaspect, the matrix can be contained within a container. In some cases,the biological sample may be fixed or immobilized on a solid support.

Solid supports of the present disclosure may be fashioned into a varietyof shapes. In certain embodiments, the solid support is substantiallyplanar. Examples of solid supports include plates such as slides,multiwell plates, flow cells, coverslips, microchips, and the like,containers such as microfuge tubes, test tubes and the like, tubing,sheets, pads, films and the like. Additionally, the solid supports maybe, for example, biological, non-biological, organic, inorganic, or acombination thereof.

As used herein, the term “solid surface” is intended to mean the surfaceof a solid support or substrate and includes any material that can serveas a solid or semi-solid foundation for attachment of a biologicalsample or other molecules such as polynucleotides, amplicons, DNA balls,other nucleic acids and/or other polymers, including biopolymers.Example types of materials comprising solid surfaces include glass,modified glass, functionalized glass, inorganic glasses, microspheres,including inert and/or magnetic particles, plastics, polysaccharides,nylon, nitrocellulose, ceramics, resins, silica, silica-based materials,carbon, metals, an optical fiber or optical fiber bundles, a variety ofpolymers other than those exemplified above and multiwell plates.Specific types of exemplary plastics include acrylics, polystyrene,copolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes and Teflon™. Specific types of exemplarysilica-based materials include silicon and various forms of modifiedsilicon.

Solid surfaces can also be varied in their shape depending on theapplication in a method described herein. For example, a solid surfaceuseful in the present disclosure can be planar, or contain regions whichare concave or convex.

Amplification

Any type of nucleic acid amplification reaction may be used to performan amplification reaction in the methods or systems described herein andgenerate an amplification product. Moreover, amplification of a nucleicacid may be linear, exponential, or a combination thereof. Non-limitingexamples of nucleic acid amplification methods include transcription(e.g., in vitro transcription), reverse transcription, primer extension,polymerase chain reaction, ligase chain reaction, helicase-dependentamplification, asymmetric amplification, rolling circle amplification,and multiple displacement amplification (MDA). In some cases, theamplified product may be DNA. In cases where a target RNA is amplified,DNA can be obtained by reverse transcription of the RNA and subsequentamplification of the DNA can be used to generate an amplified DNAproduct. In some cases, a target RNA is reverse transcribed by a reversetranscriptase to generate a cDNA. In some cases, a target DNA istranscribed by an RNA polymerase to generate an RNA. The amplified DNAproduct may be indicative of the presence of the target RNA in thebiological sample. In cases where DNA is amplified, any DNAamplification method may be employed. Non-limiting examples of DNAamplification methods include polymerase chain reaction (PCR), variantsof PCR (e.g., real-time PCR, allele-specific PCR, assembly PCR,asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR,helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR,methylation-specific PCR, miniprimer PCR, multiplex PCR, nested PCR,overlap-extension PCR, thermal asymmetric interlaced PCR, touchdownPCR), and ligase chain reaction (LCR). In some cases, DNA amplificationis linear. In some cases, DNA amplification is exponential. In somecases, DNA amplification is achieved with nested PCR, which can improvesensitivity of detecting amplified DNA products.

The amplification of nucleic acid sequences may be performed within thematrix. Methods of amplifying nucleic acids may include rolling circleamplification in situ. In certain aspects, methods of amplifying nucleicacids may include the use of PCR, such as anchor PCR, RACE PCR, or aligation chain reaction (LCR). Alternative amplification methods includebut are not limited to self-sustained sequence replication,transcriptional amplification system, Q-Beta Replicase, recursive PCR orany other nucleic acid amplification method.

The nucleic acids within the 3D matrix may be contacted with reagentsunder suitable reaction conditions sufficient to amplify the nucleicacids. The matrix may be porous to allow migration of reagents into thematrix to contact the nucleic acids. In certain aspects, nucleic acidsmay be amplified by selectively hybridizing an amplification primer toan amplification site at the 3′ end of a nucleic acid sequence usingconventional methods. Amplification primers are 6 to 100, and even up to1,000, nucleotides in length, but typically from 10 to 40 nucleotides,although oligonucleotides of different length are of use. In some cases,the amplification primer can be at least about 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or morenucleotides in length. In some cases, the amplification primer can be atleast about 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, ormore nucleotides in length. Amplification primers may hybridize to anucleic acid probe that hybridizes to a DNA molecule such that theamplification primers can be used to amplify a sequence of the nucleicacid probe. Amplification primers may be present in solution to be addedto the matrix or they may be added during formation of the matrix to bepresent therein sufficiently adjacent to nucleic acids to allow forhybridization and amplification.

A DNA polymerase can be used in an amplification reaction. Any suitableDNA polymerase may be used, including commercially available DNApolymerases. A DNA polymerase generally refers to an enzyme that iscapable of incorporating nucleotides to a strand of DNA in a templatebound fashion. Non-limiting examples of DNA polymerases include Taqpolymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENTpolymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase,Expand polymerases, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, Pho polymerase, ES4 polymerase, Tru polymerase, Tacpolymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfipolymerase, Platinum Taq polymerases, Hi-Fi polymerase, Tbr polymerase,Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase,KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, andvariants, modified products and derivatives thereof. Other enzymes canalso be used for an amplification reaction, including but not limitedto, an RNA polymerase (e.g., T7 RNA polymerase, SP6 RNA polymerase, T3RNA polymerase, etc.) and a reverse transcriptase (e.g., Avianmyeloblastosis virus (AMV) reverse transcriptase, a wild type humanimmunodeficiency virus-1 (HIV-1) reverse transcriptase, or a MoloneyMurine Leukemia Virus (M-MLV) reverse transcriptase).

Detection

The present disclosure provides methods and systems for sampleprocessing for use in nucleic acid detection. A sequence of the nucleicacid target may be identified. Various methods can be used for nucleicacid detection, including hybridization and sequencing. Nucleic aciddetection can comprise imaging the biological sample or the 3D matrixdescribed herein.

Reporter agents may be linked with nucleic acids, including amplifiedproducts, by covalent or non-covalent interactions. Non-limitingexamples of non-covalent interactions include ionic interactions, Vander Waals forces, hydrophobic interactions, hydrogen bonding, andcombinations thereof. Reporter agents may bind to initial reactants andchanges in reporter agent levels may be used to detect amplifiedproduct. Reporter agents may be detectable (or non-detectable) asnucleic acid amplification progresses. Reporter agents may be opticallydetectable. An optically-active dye (e.g., a fluorescent dye) may beused as a reporter agent. Non-limiting examples of dyes include SYBRgreen, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidiumbromide, acridines, proflavine, acridine orange, acriflavine,fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D,chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin,phenanthridines and acridines, ethidium bromide, propidium iodide,hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidiummonoazide, and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI,acridine orange, 7-AAD, actinomycin D, LDS751, hydroxystilbamidine,SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3,TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3,BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1,YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBRGreen I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45(blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25(green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59,-61, -62, -60, -63 (red), fluorescein, fluorescein isothiocyanate(FITC), tetramethyl rhodamine isothiocyanate (TRITC), rhodamine,tetramethyl rhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5,Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), Sybr Green I, SybrGreen II, Sybr Gold, CellTracker Green, 7-AAD, ethidium homodimer I,ethidium homodimer II, ethidium homodimer III, ethidium bromide,umbelliferone, eosin, green fluorescent protein, erythrosin, coumarin,methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow,cascade blue, dichlorotriazinylamine fluorescein, dansyl chloride,fluorescent lanthanide complexes such as those including europium andterbium, carboxy tetrachloro fluorescein, 5 and/or 6-carboxy fluorescein(FAM), 5- (or 6-) iodoacetamidofluorescein, 5-{[2 (and3)-5-(Acetylmercapto)-succinyl]amino}fluorescein (SAMSA-fluorescein),lissamine rhodamine B sulfonyl chloride, 5 and/or 6 carboxy rhodamine(ROX), 7-amino-methyl-coumarin, 7-Amino-4-methylcoumarin-3-acetic acid(AMCA), BODIPY fluorophores, 8-methoxypyrene-1,3,6-trisulfonic acidtrisodium salt, 3,6-Disulfonate-4-amino-naphthalimide,phycobiliproteins, AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568,594, 610, 633, 635, 647, 660, 680, 700, 750, and 790 dyes, DyLight 350,405, 488, 550, 594, 633, 650, 680, 755, and 800 dyes, or otherfluorophores.

In some embodiments, a reporter agent may be a sequence-specificoligonucleotide probe that is optically active when hybridized with anucleic acid target or derivative thereof (e.g., an amplified product).A probe may be linked to any of the optically-active reporter agents(e.g., dyes) described herein and may also include a quencher capable ofblocking the optical activity of an associated dye. Non-limitingexamples of probes that may be useful used as reporter agents includeTaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes.

In some aspects, the method for determining the nucleic acid sequence ofa target nucleic acid molecule includes sequencing. In some aspects,sequencing by synthesis, sequencing by ligation or sequencing byhybridization is used for determining the nucleic acid sequence of atarget nucleic acid molecule. As disclosed herein, various amplificationmethods can be employed to generate larger quantities, particularly oflimited nucleic acid samples, prior to sequencing. For example, theamplification methods can produce a targeted library of amplicons.

For sequencing by ligation, labeled nucleic acid fragments may behybridized and identified to determine the sequence of a target nucleicacid molecule. For sequencing by synthesis (SBS), labeled nucleotidescan be used to determine the sequence of a target nucleic acid molecule.A target nucleic acid molecule can be hybridized with a primer andincubated in the presence of a polymerase and a labeled nucleotidecontaining a blocking group. The primer can be extended such that thelabeled nucleotide is incorporated. The presence of the blocking groupmay permit the incorporation of a single nucleotide. The presence of thelabel can permit identification of the incorporated nucleotide. As usedherein, a label can be any optically active dye described herein. Eithersingle bases can be added or, alternatively, all four bases can be addedsimultaneously, particularly when each base is associated with adistinguishable label. After identifying the incorporated nucleotide byits corresponding label, both the label and the blocking group can beremoved, thereby allowing a subsequent round of incorporation andidentification. Thus, cleavable linkers can link the label to the base.Examples of cleavable linker include, but are not limited to, peptidelinkers. Additionally, a removable blocking group may be used so thatmultiple rounds of identification can be performed, thereby permittingidentification of at least a portion of the target nucleic acidsequence. The compositions and methods disclosed herein are useful forsuch an SBS approach. In addition, the compositions and methods can beuseful for sequencing from a solid support (e.g., an array or a samplewithin a 3D matrix as described herein), where multiple sequences can be“read” simultaneously from multiple positions on the solid support sinceeach nucleotide at each position can be identified based on itsidentifiable label. Example methods are described in US 2009/0088327; US2010/0028885; and US 2009/0325172, each of which is incorporated hereinby reference.

RNA Degradation

The methods and systems described herein may use a probe for targetcapture or detection. For example, the probe may be a padlock probe. Inthe padlock probe capture method, a ligation reaction of a DNA-DNAduplex template formed between a cDNA molecule and the DNA padlock probecan be generally more efficient than a ligation reaction of a DNA-RNA“hybrid” duplex template formed between an RNA molecule and DNA padlockprobe, due to the enhanced efficiency of enzymatic ligation betweenDNA-DNA duplex templates compared to DNA-RNA hybrid duplex templates.Therefore, all or part of a target RNA molecule may be first convertedinto a cDNA molecule, as by reverse transcription, prior to the captureof target molecule by the padlock probe. In some cases, the padlockprobe may be extended via a DNA polymerization reaction, using the cDNAmolecule as a template, until the 3′ and 5′ ends of the padlock probeare proximal for the ligation reaction to occur.

After converting all or part of a target RNA molecule into a cDNAmolecule, the RNA molecule may be degraded. In some cases, the RNA maybe degraded using a non-ribonuclease enzyme with RNA catalytic cleavageactivity. In some other cases, the RNA may be degraded by chemicaldecomposition under conditions wherein cDNA can remain substantiallychemically stable.

In some cases, a target RNA molecule may be directly captured by (e.g.,hybridized to) a DNA padlock probe without reverse transcription step.In this case, the padlock probe may be extended using the target RNAmolecule as a template or may be circularized while the padlock probehybridized to the target RNA molecule. The target RNA molecule can thenbe degraded after circularization of the padlock probe.

Reverse Transcriptases

The RNA molecule may be enzymatically digested via a non-ribonucleaseenzyme. According to a certain embodiment, the enzymatic digestion ofthe RNA molecule can be catalyzed by a reverse transcriptase. Certainreverse transcriptases may possess ribonuclease activity, including butnot limited to RNaseH activity. Certain reverse transcriptases maypossess the activity of specifically digesting RNA within an RNA-DNAhybrid duplex. For example, Avian myeloblastosis virus (AMV) reversetranscriptase possesses an intrinsic RNase H activity, which can degradethe RNA strand of an RNA/DNA hybrid. Other reverse transcriptasesexhibiting ribonuclease activity or RNA catalytic cleavage activityinclude but are not limited to the wild type HIV-1 and M-MLV reversetranscriptases.

Most reverse transcriptases may not possess ribonuclease activity or RNAcatalytic cleavage activity, thereby enhancing the rate of full-lengthcDNA synthesis. For example, the most popular variant of M-MLV RT is theM-MLV RT RNase H-point mutant, which has a single amino acidsubstitution that dramatically reduces RNase H activity. However, forthe purpose of padlock probe assay as described herein, the reversetranscription reaction may be primed, either specifically by using asubstantially complementary primer, or non-specifically, by using a poolof degenerate primers, proximal to the padlock probe capture site alongthe RNA/cDNA molecule, in which case full-length cDNA synthesis may notbe needed for efficient padlock probe capture.

Using a reverse transcriptase to degrade or digest RNA may provide costand time savings compared to using a separate enzymatic reaction for RNAdigestion. The methods described herein may also reduce the number ofcomponents and/or steps in a padlock probe assay. Furthermore, usingthis strategy the ribonuclease activity of the reverse transcriptase maybe modulated by varying the composition of the reaction buffer to enablea multi-phase reaction comprising a first phase of efficient reversetranscription, and a subsequent phase of efficient RNA digestion.Aspects of reaction buffer composition modulating ribonuclease activitymay include ribonuclease inhibitors, including organic chemicals andpolypeptides, cofactors including metal ions, such as Mg²⁺, Mn²⁺, Na¹⁺,ATP, NADPH, etc. For example, reverse transcription by HIV-1 RTase mayproceed efficiently under low concentrations of Mg2+ ions, despitestrongly reduced intrinsic polymerase activity, by decreasing thedegradation of RNA template by the RNase activity, while subsequentlyincreasing the concentration of Mg^(2|) can increase the ribonucleaseactivity to liberate the cDNA from the hybrid duplex.

DNA Binding Proteins

According to another aspect of the present disclosure, the RNA can bedigested by another non-ribonuclease enzyme possessing ribonucleaseactivity or RNA catalytic cleavage activity. In a particular embodiment,a DNA binding protein with ribonuclease activity or RNA catalyticcleavage activity may be used for dual purposes. One of the purposes maybe digestion of the RNA molecule. One such other purpose may bestabilization of the single-stranded cDNA molecule, such as by asingle-stranded DNA (ssDNA) binding protein. The DNA binding protein mayalso promote hybridization between the cDNA molecule and the DNA padlockprobe molecule. For example, the DNA binding protein Sso7d can be usedto promote the annealing of complementary DNA strands above the meltingpoint of the duplex, and may also be used to degrade RNA since Sso7possesses ribonuclease activity.

Chemical Decomposition

The RNA can be subjected to chemical decomposition under conditionswherein cDNA remains substantially chemically stable. RNA hydrolysis maybe a reaction in which a phosphodiester bond in the sugar-phosphatebackbone of RNA is broken, cleaving the RNA molecule. RNA can besusceptible to this base-catalyzed hydrolysis because the ribose sugarin RNA has a hydroxyl group at the 2′ position. This feature may makeRNA chemically unstable compared to DNA, which does not have this 2′ OHgroup and thus may not be susceptible to base-catalyzed hydrolysis.Chemical decomposition of RNA, such as by hydrolysis, may occur under avariety of conditions under which DNA including single-stranded cDNA canremain substantially chemically stable. RNA may be thermolabile andsusceptible to metal-catalyzed degradation. Under normal conditions RNAhydrolysis can occur at a low frequency, but RNA hydrolysis can beaccelerated under certain conditions, for example, acidic pH, alkalinepH, high temperatures, in the presence of divalent cations, and in thepresence of heavy metal ions.

A buffer may be used to provide a condition for chemical decompositionof the RNA. For example, a buffer can comprise Tris HCL with a pH of atleast about 7, 7.5, 8, or more. The final concentration of Tris HCL canbe at least about 20, 30, 40, 50, or more mM. The buffer can furthercomprise MgCL₂ with a final concentration of at least about 15, 20, 25,30, 35, 40, 45, 50, or more mM. In some cases, the buffer comprises 50mM Tris HCL with a pH of 7.5-8 and 20-50 mM MgCL₂. For another example,a buffer can comprise sodium borate with a pH of at least about 7, 7.5,8, or more. The final concentration of sodium borate can be at leastabout 20, 30, 40, 50, or more mM. The buffer can further comprise MgCL₂with a final concentration of at least about 15, 20, 25, 30, 35, 40, 45,50, or more mM. In some cases, the buffer comprises 50 mM Sodium Borateat pH 7.5-8 with 20-50 mM MgCl₂. The divalent cation may not be limitedto Mg²⁺, and can be other types of divalent cations such as Mn²⁺.

The buffer used for chemical decomposition of RNAs may also catalyze aclick reaction between click reactive groups. In some cases, a molecule(e.g., a target, a primer, a probe, or a molecule derived from a targetsuch as cDNA) may be tethered via a click reaction to a click reactivegroup functionalized hydrogel matrix (e.g., click gel). For example, the5′azidomethyl-dUTP can be incorporated into cDNA and then immobilized tothe hydrogel matrix functionalized with alkyne groups. Various clickreactions may be used. The buffer described herein can both catalyze thefunctional immobilization linkage between the molecule and the matrixand hydrolyze the RNA molecule, liberating the DNA from the DNA-RNAhybrid duplex. Using the buffer having both functions in chemicaldeposition and click reaction catalyzation may provide an improvement inworkflow efficiency, reduce workflow time, and reduce the number ofassay reagents. For example, the buffer may be a Cu(I)-catalyzedalkyne-azide cycloaddition (abbreviated as CUAAC) click reactioncatalyzing buffer, which catalyzes the alkyne-azide bond in the clickreaction. The buffer can comprise 1-25 mM copper (II) sulfate solution,1-50 mM Tris((1-hydroxy-propyl-1H-1,2,3-triazol-4-yl)methyl)amine(abbreviated as THPTA) solution, 5-100 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (abbreviated asHEPES) buffer, and 5-100 mM L-ascorbic acid. In some cases, the copper(II) sulfate may be at a final concentration of at least about 1, 5, 10,15, 20, 25, or more mM. In some cases, the THPTA may be at a finalconcentration of at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, or more mM. In some cases, the HEPES may be at a final concentrationof at least about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, or more mM. In some cases, the L-ascorbicacid may be at a final concentration of at least about 2, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or moremM. Optionally, the buffer may be degassed or argon bubbled to removedissolved oxygen prior to use.

Using the RNA degradation methods described herein may provide cost andtime savings and improve the efficiency of the liberation of the cDNAfrom the hybrid duplex. The chemical decomposition of RNA can be fasterand more efficient than enzymatic digestion. Moreover, the chemicalnon-enzymatic reagents may be cheaper, easier to synthesize, easier tostore, and have longer shelf life compared to reagents used in enzymaticreactions.

Nucleic Acid Immobilization in a 3D Matrix

To preserve the spatial information associated with each RNA moleculebeing detected in the padlock probe assay, the cDNA molecule within thebiological sample (e.g., cell and tissue) may be spatially immobilizedat the original position of the RNA molecule. Similarly, in the case ofdirectly capturing target RNA or DNA molecule by a probe (e.g., apadlock probe), the probe may be spatially immobilized at the originalposition of the target RNA or DNA molecule. The spatial origin of thetarget RNA or DNA molecule itself may be typically preserved by theformation of chemical or physical crosslinks between the target RNA orDNA molecule and the naturally-occurring 3D matrix of biomolecules(e.g., proteins) within the biological sample. The chemical or physicalcrosslinks can be formed by temperature, electromagnetic radiation(e.g., microwave), or chemicals such as formaldehyde and glutaraldehydeand others within the cell and tissue. The naturally-occurring 3D matrixmay be formed by crosslinking endogenous or native biomolecules, such asproteins and nucleic acids, within a cell or tissue. The spatial originof the RNA molecule may also be preserved by the formation of chemicalor physical crosslinks between the RNA molecule and other natural orsynthetic components added to the sample to supplement or replace nativecellular components for the purpose of immobilizing the cDNA. Forexample, a synthetic 3D matrix may be formed in situ throughout the celland tissue sample in order to preserve a spatial position of the RNAmolecule or DNA molecule. The synthetic 3D matrix can be a hydrogelmatrix. For example, the 3D matrix may be composed of polyacrylamide orpolyethylene glycol (PEG). During cDNA synthesis, the spatial locationof the cDNA can be preserved via hydrogen bonding between the RNA andcDNA within the hybrid duplex (“hybridization”). However, the spatiallocation of the cDNA independent of the RNA molecule can be preserved,for example, to avoid the loss of spatial position after liberation ofthe cDNA from the hybrid RNA-DNA duplex.

The functional moiety can bind to or react with a cell or cellularcomponent. The affinity binding group can bind to a cell or cellularcomponent. The functional moiety may comprise at least one nucleotidemodified with biotin, an amine group, a lower alkylamine group, anacetyl group, DMTO, fluoroscein, a thiol group, or acridine. In the casea synthetic 3D matrix is provided for cDNA cross-linking, the sample maybe immersed in a gel solution which upon polymerization can give rise toa gel matrix to which the cDNA molecule or target RNA molecule can beattached. As described herein, approaches to immobilize nucleic acidmolecules in a matrix are provided.

Immobilization of cDNA Molecules

Approaches to immobilizing the cDNA molecule independently of itshybridization to the RNA are provided. Generically, these advances canfall into three categories: a) the in situ polymerized component of thecDNA can comprise the functional moiety for immobilization; b) thefunctional moiety for immobilization can react with a pre-existing (atthe time of reverse transcription) in situ matrix of non-cellularorigin, i.e. a synthetic or exogenous matrix; and c) the functionalimmobilization moiety can be associated with the reverse transcriptionprimer by hydrogen bonding via DNA hybridization.

According to the first aspect, the in situ polymerized component of thecDNA can comprise the functional moiety for immobilization, rather thanthe primer from which reverse transcription is initiated, or primed.

The cDNA molecule may be functionalized during the reverse transcriptionreaction, such as by adding nucleotide triphosphate analogs comprisingfunctional moieties for immobilization. Such nucleotide triphosphateanalogs include, but are not limited to, amino-allyl dUTP,5-TCO-PEG4-dUTP, C8-Alkyne-dUTP, 5-Azidomethyl-dUTP, 5-Vinyl-dUTP,5-Ethynyl dUTP, and other nucleotide triphosphate analogs comprising afunctional moiety for cDNA immobilization by cross-linking, or forming achemical bond between the cDNA and in situ matrix, cellular orsynthetic. Furthermore, the in situ matrix, cellular or synthetic, maycontain or be made to contain chemical moieties (e.g., reactive groups)that can react with the functional moieties in the cDNA throughfunctionalization reactions. For example, amino-allyl dUTP may becross-linked to endogenous free amine groups present in proteins andother biomolecules present within the endogenous or exogenous cellularmatrix, or present in a modified synthetic hydrogel matrix, such as anamine-functionalized polyacrylamide hydrogel formed by copolymerizationof polyacrylamide and N-(3-aminopropyl)-methacrylamide; likewisenucleoside analogs containing azide functional moieties may becross-linked to a synthetic hydrogel matrix comprising alkyne functionalmoieties, such as that formed by copolymerization of acrylamide andpropargyl acrylamide.

The cDNA molecule may be functionalized with moieties for immobilizationsubsequent to reverse transcription. Mechanisms for post-synthesis cDNAfunctionalization may include a variety of biochemical and chemicalmethods. These include, but are not limited to, use of a ligationreaction to conjugate an oligonucleotide bearing a functional moiety forimmobilization to the cDNA molecule, use of a DNA polymerizationreaction to add templated or un-templated bases to the cDNA, as in theprocess of A-tailing by Taq polymerize, or by using the reactionsmediated by DNA end-repair mechanisms. Alternatively, a chemical methodof DNA chemical functionalization may be used to conjugate functionalmoieties for immobilization. For example, Label-IT Amine and Label-X arebifunctional reagents that can react with nucleic acids via a nitrogenmustard alkylation mechanism for the purpose of conjugating free amineor acryloyl groups to the nucleic acid, which can be used for thepurpose of immobilization to the matrix. Other chemistries, includingbut not limited to, DNA alkylation and oxymercuration, can providemechanisms for functionalizing DNA.

The approaches for cDNA immobilization provided herein provide severaladvantages. For example, using the approaches provided herein,unmodified DNA oligonucleotides may be used as reverse transcriptionprimers, simplifying the manufacturing of these reagents and reducingassay cost. Furthermore, by restricting cross-linking functionalmoieties to newly polymerized components of the cDNA, residual reversetranscription primers which have not participated in a reversetranscription reaction may be easily washed out of the sample and cannotparticipate in the cDNA immobilization mechanism, which may reducebackground signals or improve downstream reaction yields. In addition,using a separate reaction to functionalize the cDNA with functionalmoieties after reverse transcription may simplify experimentalworkflows. For example, a DNA functionalization mechanism can be used tosimultaneously functionalize cDNA and genomic DNA with functionalmoieties in a single reaction, facilitating multi-omic in situ assayssuch as fluorescent in situ sequencing (FISSEQ).

The functional moiety for immobilization may react or cross-link with apre-existing (at the time of reverse transcription) in situ matrix ofnon-cellular origin, i.e. a synthetic or exogenous matrix. According tothis aspect, a synthetic chemical matrix bearing functional moieties forcDNA immobilization may be formed in situ prior to reversetranscription. Suitable functional moieties include but are not limitedto amine and click functional groups. After synthesis of the cDNAmolecule, functional moieties present in the cDNA can cross-link withcomplementary functional moieties with the matrix. By using a pre-formedin situ synthetic matrix for immobilization, all or part of the naturalbiological matrix of the sample may be degraded, removed, or chemicallymodified, thereby enhancing the reaction properties, including yield, ofthe reverse transcription or other reactions.

Modification of Primers

The reverse transcription primer itself may bear no functional moietiesfor immobilization, but instead may be associated with one or morefunctional moieties for cDNA immobilization via hydrogen bonding. Thisapproach may enable the use of unmodified reverse transcription primers,thereby simplifying and reducing the cost of assay manufacturing. Forexample, the primers may bear a functional domain, such as a commondomain among a plurality of reverse transcription primers, comprising atethering oligonucleotide hybridization site, and the tetheringoligonucleotide can bear the functional moiety for cDNA immobilization.Furthermore, the tethering oligonucleotide may bear additional features,such as partial complementarity to the domains responsible for bindingthe RNA and initiating reverse transcription. The tetheringoligonucleotide may bear additional sequence used for enhancing thespecificity of the reverse transcription primer-cDNA annealing reactionvia competitive hybridization mechanisms.

In some other cases, a primer (e.g., a reverse transcription primer) maycomprise a functional moiety for immobilization onto the 3D matrix. Forexample, a primer may not hybridize to a tethering oligonucleotidehaving a functional moiety, but instead can be directly covalentlylinked to a functional moiety for immobilization.

Modification of Padlock Probes

The padlock probe may comprise functional moieties for immobilization tothe in situ matrix, either directly or indirectly, as via a hybridizedoligonucleotide. For example, a tethering oligonucleotide hybridized tothe backbone of the padlock probe, e.g., outside the domains responsiblefor hybridizing to the target cDNA molecule, may serve as a rollingcircle amplification primer, thereby serving to tether the padlock probemolecule (and cDNA molecule) via DNA hybridization prior to rollingcircle amplification, and subsequently serving to tether the rollingcircle amplicon (i.e., rolony) after rolling circle amplification forthe purpose of preserving the spatial information associated with theoriginal RNA molecule, cognate cDNA molecule, padlock probe, and rolony.

In some cases, the efficiency, or reaction yield, of establishing thefunctional immobilization linkage between the target molecule, probe,cDNA, padlock probe, or amplicon to the 3D matrix can be enhanced by thepresence of more than one functional moiety within a molecule to betethered. In the case of functional moiety incorporation into the cDNAduring reverse transcription, this is accomplished by titrating theamount of functionally modified dNTP in the mix. In some embodiments,where a tethering oligonucleotide is used to indirectly immobilize thetarget or probe by hydrogen bonding provided by DNA hybridization,tethering oligonucleotides bearing multiple functional moieties can besynthesized. For example, tethering oligonucleotide bearing multiplefunctional moieties can be synthesized by incorporation of internalamines during chemical DNA synthesis, and subsequent conversion (e.g.,en masse conversion) of amines to azide, acryloyl, or other functionalmoieties. The conversion can be achieved by amine-reactive chemicalgroups such as NHS-esters. The inefficiency of single-molecule tetheringto the 3D matrix using a single tethering moiety may result from eitherintrinsic rate of the immobilization reaction (i.e., the chance anacryloyl is incorporated into the in situ polymerizing matrix, or thechance of an azide coming into proximity to the alkyne present in theclick gel), or due to “non-ideal” in situ matrix synthesis processes.For example, the “non-ideal” in situ matrix synthesis process can referto the situation where the single molecule is conjugated to a polymer,but that polymer is not itself stably linked into the larger emergent 3Dnetwork architecture generated during formation of the 3D matrix, whichmerely increases the molecular weight of the molecule but still allowsthe molecule and the conjugated polymer thereof to diffuse from the 3Dmatrix. The presence of a plurality of immobilization moieties canmitigate both of these mechanisms by increasing the chance a singlemolecule is incorporated as the sum total or product of likelihood ofeach moiety incorporation reaction, and also by providing the potentialfor linkage to multiple polymer chains within the 3D matrix. Thesemultiple functional moieties may be in close proximity, oralternatively, spaced at regular or irregular intervals within themolecule. For example, multiple functional moieties can be presentconsecutively within a nucleic acid molecule. For example, multiplefunctional moieties can be separated by at least 1, 2, 3, 4, 5, 6, 7, ormore nucleotides within a nucleic acid molecule.

Methods for Sample Processing

In an aspect, the present disclosure provides a method foridentification of a nucleic acid sequence in a biological sample. Themethod may comprise providing the biological sample comprising aribonucleic acid (RNA) molecule hybridized to a deoxyribonucleic acidmolecule (DNA) in a three-dimensional (3D) matrix. The RNA molecule maycomprise the nucleic acid sequence. Next, a reverse transcriptase or afunctional derivative thereof may be used to degrade or digest at leasta portion of the RNA molecule hybridized to the DNA molecule. The DNAmolecule may comprise an additional nucleic acid sequence that is areverse complement of the nucleic acid sequence. Next, the additionalnucleic acid sequence in the biological sample may be detected, therebyidentifying the nucleic acid sequence.

The DNA molecule may be a complementary deoxyribonucleic acid (cDNA)molecule. For example, the DNA molecule may be reverse transcribed fromthe RNA molecule. The cDNA molecule may be reverse transcribed from theRNA molecule and amplified (e.g., by polymerase chain reaction (PCR))through one or more rounds of amplification to generate one or morecopies of a sequence of the cDNA molecule.

In some cases, an additional reverse transcriptase or functionalderivative thereof may be used to reverse transcribe the RNA molecule togenerate the DNA molecule hybridized to the RNA molecule in thebiological sample. As an alternative, the reverse transcriptase orfunctional derivative thereof, which was used to degrade or digest atleast a portion of the RNA molecule, may be used to initially reversetranscribe the RNA molecule to generate the DNA molecule hybridized tothe RNA molecule in the biological sample.

The DNA molecule may be immobilized to the 3D matrix. For example, theDNA molecule may be covalently immobilized to the 3D matrix (e.g., bycross-linking, such as using disulfide bonds). The DNA molecule maycomprise a functional moiety, and the DNA molecule may be immobilized tothe 3D matrix via the functional moiety. Examples of functional moietyinclude but are not limited to an amine, acrydite, alkyne, biotin,azide, and thiol. The RNA molecule may be immobilized to the 3D matrix.The RNA molecule may comprise a functional moiety, and the RNA moleculemay be immobilized to the 3D matrix via the functional moiety. Forexample, the RNA molecule may be modified with LabelX which can be usedto covalently attach the RNA molecule to the 3D matrix. The compoundLabelX can be synthesized from Acryloyl-X SE(6-((acryloyl)amino)hexanoic acid, succinimidyl ester) and Label-ITamine (MirusBio) using NHS-ester chemistry and can react with RNA, forexample, with the N7 position of guanines. The LabelX reagent can enabledevelopment of other attachment chemistries beside free-radicalpolymerization of acryloyl into polyacrylamide. For example, anNHS-ester-azide compound may be conjugated to Label-IT Amine to create anew linker capable of tethering nucleic acids into a PEG-click hydrogelmatrix.

In some cases, a matrix-forming material may be used to form the 3Dmatrix. The matrix forming material may be polymerizable monomers orpolymers, or cross-linkable polymers. The matrix forming material may bepolyacrylamide, acrylamide monomers, cellulose, alginate, polyamide,agarose, dextran, or polyethylene glycol. The matrix forming materialscan form a matrix by polymerization and/or crosslinking of the matrixforming materials using methods specific for the matrix formingmaterials and methods, reagents and conditions. The matrix formingmaterial may form a polymeric matrix. The matrix forming material mayform a polyelectrolyte gel. The matrix forming material may form ahydrogel gel matrix.

In some cases, the cDNA molecule may be contacted with a probe. The cDNAmolecule may hybridize to the probe. The probe may comprise a functionalmoiety. The probe may be immobilized to the 3D matrix via the functionalmoiety. The cDNA molecule can be indirectly immobilized to the 3D matrixvia the functional moiety by hybridizing to the probe. The functionalmoiety can bind to or react with a cell or cellular component, or can bean affinity binding group capable of binding to a cell or cellularcomponent. The functional moiety may comprise at least one nucleotidemodified with biotin, an amine group, a lower alkylamine group, anacetyl group, DMTO, fluoroscein, a thiol group, or acridine. The probemay be a padlock probe, wherein the padlock probe comprises 5′ and 3′terminal regions complementary to the cDNA molecule. The 5′ and 3′terminal regions of the padlock probe can be hybridized to the cDNAmolecule. After hybridizing with the cDNA molecule, the 5′ and 3′ endscan be brought into juxtaposition. The two ends of the padlock probe maybe contiguous or separated by a gap region of the cDNA molecule. The gapregion may be of various lengths. For example, the gap region cancomprise at least 1, at least 5, at least 10, at least 15, at least 20,at least 25, at least 30, at least 35, at least 40, at least 45, or atleast 50 nucleotide(s). For another example, the gap region can comprisefrom 2 to 10, from 10 to 20, from 20 to 50, from 50 to 100, from 100 to150, from 150 to 200, from 200 to 300, from 300 to 400, or from 400 to500 nucleotides. A circularized padlock probe can be generated byligating two ends of the padlock probe together. In the cases where thetwo ends are contiguous, the two ends can be ligated directly. In thecases where the two ends are separated by a gap region, the gap regioncan be filled in before ligation. The gap region can be filled in byincorporating one or more nucleotides in an extension reaction, forexample, by extending from the 3′ end of the two ends. The extensionreaction can be performed by a polymerase. The extension reaction can becarried out using the cDNA molecule as a template since the 5′ end and3′ end of the padlock probe are hybridized to the cDNA molecule, therebycapturing sequence information of the cDNA molecule into the padlockprobe. The gap region can also be filled in by hybridizing an additionalnucleotide or an additional oligonucleotide sequence to the gap region.The length of the additional oligonucleotide sequence can be determinedbased on the length of the gap region. For example, the additionaloligonucleotide sequence can be of the same length as the gap regionsuch that after hybridizing with the gap region, the 5′ end of theadditional oligonucleotide sequence is adjacent to the 3′ end of thepadlock probe and the 3′ end of the additional oligonucleotide sequenceis adjacent to the 5′ end of the padlock probe. For example, theadditional oligonucleotide sequence can comprise from 2 to 10, from 10to 20, from 20 to 50, from 50 to 100, from 100 to 150, from 150 to 200,from 200 to 300, from 300 to 400, or from 400 to 500 nucleotides. Thecircularized padlock probe can then be generated by ligating the ends ofthe additional oligonucleotide sequence with the ends of the padlockprobe.

The circularized padlock probe may be further subjected to rollingcircle amplification (RCA) to generate an amplification product of asequence of the circularized padlock probe. The amplification productcan comprise a nucleic acid sequence corresponding to the nucleic acidsequence of the RNA molecule. The nucleic acid sequence of theamplification product can be detected, thereby identifying the nucleicacid sequence of the RNA molecule. Different detection methods may beused for nucleic acid detection including sequencing and hybridization.Examples of sequencing methods include sequencing by synthesis (SBS),sequencing by ligation (SBL), and sequencing by hybridization (SBH).

The reverse transcriptase or functional derivative thereof may have RNAcatalytic cleavage activity. For example, the reverse transcriptase orfunctional derivative thereof may have ribonuclease (RNase) activity.The reverse transcriptase or functional derivative thereof may cleaveRNA of a RNA/DNA duplex. The additional reverse transcriptase orfunctional derivative thereof may also have RNA catalytic cleavageactivity. The reverse transcriptase or the additional reversetranscriptase may be an Avian myeloblastosis virus (AMV) reversetranscriptase, a wild type human immunodeficiency virus-1 (HIV-1)reverse transcriptase, or a Moloney Murine Leukemia Virus (M-MLV)reverse transcriptase.

To reverse transcribe the RNA molecule, a reverse transcription primermay be hybridized to the RNA molecule. The reverse transcription primermay be hybridizable to the 5′ terminal region of the padlock probe. Thereverse transcription primer may not be hybridizable to the 5′ terminalregion of the padlock probe. In the situation where the reversetranscription primer is not hybridizable to the 5′ terminal region ofthe padlock probe, the region of the generated cDNA molecule that iscomplementary to the 5′ terminal region of the padlock probe may bedownstream of the reverse transcription primer by a distance of at least1, at least 5, at least 10, at least 50, at least 100, at least 200, atleast 500, or more nucleotides. As used herein, the distance between aregion A (e.g., the region of the cDNA molecule complementary to the 5′terminal region of the padlock probe) and a region B (e.g., reversetranscription primer) of the same nucleic acid strand, wherein theregion A is downstream of the region B, refers to the number ofnucleotides between the 5′ end of region A and the 3′ end of the regionB. As used herein, downstream refers to the direction from 5′ end to 3′end of a nucleic acid strand.

The reverse transcription primer may comprise a functional moiety. Thereverse transcription primer or an extension product thereof (e.g., cDNAmolecule) can be immobilized to the 3D matrix via the function moiety.Examples of functional moiety include but are not limited to an amine,acrydite, alkyne, biotin, azide, and thiol.

The biological sample may comprise a plurality of target molecules. Forexample, the target molecules can be DNA molecules, RNA molecules, orprotein molecules. The target molecules may have a relative 3D spatialrelationship in the 3D matrix. In some cases, the biological samplecomprises a plurality of RNA molecules. The plurality of RNA moleculesmay have a relative 3D spatial relationship in the 3D matrix.

In some cases, the RNA molecule or a portion thereof may be degraded ordigested by the reverse transcriptase under a first set of conditionsand the RNA molecule may be reverse transcribed by the same reversetranscriptase or an additional reverse transcriptase under a second setof conditions. The first set of conditions can be different than thesecond set of conditions. The first set of conditions may be the same asthe second set of conditions. The first set of conditions or the secondset of conditions can be selected from the group consisting of pH,temperature, cofactor concentration, and cation concentration. Examplesof cofactor or cation include but are not limited to Mg²⁺, Mn²⁺, Na⁺,ATP, and NADPH. The second set of conditions may inhibit RNase activityof the reverse transcriptase that is used to reverse transcribe the RNAmolecule. For example, the second set of conditions may comprise anRNase inhibitor. The RNase inhibitor may be a small molecule inhibitoror a polypeptide.

The cDNA molecule may comprise a functional moiety. The cDNA moleculecan be immobilized to the 3D matrix via the functional moiety. Thefunctional moiety can be covalently crosslinked, copolymerize with orotherwise non-covalently bound to the matrix. The functional moiety canreact with a crosslinker. The functional moiety can be part of aligand-ligand binding pair. DNTP or dUTP can be modified with thefunctional group, so that the function moiety can be introduced into theDNA during amplification. Examples of functional moiety include but arenot limited to an amine, acrydite, alkyne, biotin, azide, and thiol. Inthe case of crosslinking, the functional moiety can be crosslinked tomodified dNTP or dUTP or both. Examples of crosslinker reactive groupsinclude but are not limited to imidoester (DMP), succinimide ester(NHS), maleimide (Sulfo-SMCC), carbodiimide (DCC, EDC) and phenyl azide.Crosslinkers within the scope of the present disclosure may include aspacer moiety. Such spacer moieties may be functionalized. Such spacermoieties may be chemically stable. Such spacer moieties may be ofsufficient length to allow amplification of the nucleic acid bound tothe matrix. Examples of spacer moieties include but are not limited topolyethylene glycol, carbon spacers, photo-cleavable spacers and otherspacers and the like.

In another aspect, the present disclosure provides a method foridentification of a nucleic acid sequence in a biological sample. Themethod may comprise providing the biological sample comprising a RNAmolecule hybridized to a DNA in a 3D matrix. The RNA molecule maycomprise the nucleic acid sequence. Next, a DNA binding protein may beused to degrade or digest at least a portion of the RNA moleculehybridized to the DNA molecule. The DNA binding protein may not be areverse transcriptase, a ribonuclease, or both. The DNA molecule maycomprise an additional nucleic acid sequence that is a reversecomplement of said nucleic acid sequence. Next, the additional nucleicacid sequence in the biological sample may be detected, therebyidentifying the nucleic acid sequence. In some cases, a reversetranscriptase may be used to reverse transcribe the RNA molecule togenerate the DNA molecule hybridized to the RNA molecule in thebiological sample.

The DNA binding protein or functional derivative thereof may have RNAcatalytic cleavage activity. For example, the DNA binding protein orfunctional derivative thereof may have ribonuclease (RNase) activity.The DNA binding protein or functional derivative thereof may cleave theRNA of a RNA/DNA duplex. The DNA binding protein may stabilize the DNAmolecule. The DNA binding protein may stabilize the DNA of a RNA/DNAduplex. The DNA binding protein may increase a melting temperature ofthe DNA molecule. The DNA binding protein may stabilize the DNA moleculeby increasing the melting temperature of the DNA molecule. In somecases, the DNA binding protein is Sso7d.

In another aspect, the present disclosure provides a method foridentification of a nucleic acid sequence in a biological sample. Themethod may comprise providing the biological sample comprising a RNAmolecule hybridized to a DNA molecule in a 3D matrix. The RNA moleculemay comprise the nucleic acid sequence. Next, at least a portion of saidRNA molecule hybridized to the DNA molecule may be non-enzymaticallydegraded. The DNA molecule may comprise an additional nucleic acidsequence that is a reverse complement of the nucleic acid sequence.Next, the DNA molecule may be contacted with a probe. The DNA moleculemay hybridize with the probe upon contacting with the probe. Next, asequence of the probe or a derivative thereof may be detected, therebyidentifying the nucleic acid sequence of the RNA molecule.

The RNA molecule may be subjected to chemical degradation. The RNAmolecule may be degraded or hydrolyzed in the absence of an enzyme. Forexample, RNA can be susceptible to base-catalyzed hydrolysis because theribose sugar in RNA has a hydroxyl group at the 2′ position. RNAhydrolysis can occur when the deprotonated 2′ OH of the ribose, actingas a nucleophile, attacks the adjacent phosphorus in the phosphodiesterbond of the sugar-phosphate backbone of the RNA. As used herein, RNAhydrolysis refers to a reaction in which a phosphodiester bond in thesugar-phosphate backbone of RNA is broken, cleaving the RNA molecule.Inorganic and organic compounds may be used to degrade or cleave RNA. Insome cases, a metal complex may be used to degrade or cleave RNA. Insome cases, a heavy metal ion may be used to degrade or cleave RNA. Boththe transesterification step, where a 2′,3′-cyclic phosphate may beformed with concomitant cleavage of RNA, and the hydrolysis step, wherethe 2′,3′-cyclic phosphate may be converted to a phosphate monoester,can be catalyzed by inorganic or organic compounds.

The chemical degradation may be performed under a neutral to alkaline pHcondition. For example, the pH may have a value of at least 6, at least6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, atleast 9.5, at least 10, at least 10.5, at least 11, at least 11.5, atleast 12, at least 12.5, at least 13, at least 13.5, or 14. For anotherexample, the pH may be from 6 to 7, from 7 to 8, from 8 to 9, from 9 to10, from 10 to 11, from 11 to 12, from 12 to 13, or from 13 to 14. Thechemical degradation may be performed under an acidic to neutral pHcondition. For example, the pH may have a value of 0, at most 1, at most2, at most 3, at most 4, at most 5, at most 6, or at most 7. For anotherexample, the pH may be from 0 to 1, from 1 to 2, from 2 to 3, from 3 to4, from 4 to 5, from 5 to 6, or from 6 to 7. The chemical degradationmay be performed under a temperature condition in which RNA is unstable.For example, the temperature condition suitable for RNA degradation maybe at least 5° C., at least 10° C., at least 20° C., at least 30° C., atleast 40° C., at least 50° C., at least 60° C., at least 70° C., atleast 80° C., at least 90° C., at least 100° C., or more. For anotherexample, the temperature condition suitable for RNA degradation may befrom 5° C. to 10° C., from 10° C. to 20° C., from 20° C. to 30° C., from30 to 40° C., from 40 to 50° C., from 50 to 60° C., from 60 to 70° C.,from 70 to 80° C., from 80 to 90° C., or from 90 to 100° C. The chemicaldegradation may be performed in the presence of a heavy metal ion. Theterm heavy metal refers to any metallic chemical element that has arelatively high density and may be toxic or poisonous at lowconcentrations. The heavy metal ion may be part of a metal complex. Asused herein, the metal complex refers to a macrocyclic complex formed bythe union of a central metal ion with a non-metallic ion or molecule.The metal ion can comprise copper, zinc, cobalt, nickel, palladium,lead, iridium, manganese, iron, molybdenum, vanadium, titanium,ruthenium, bismuth, cadmium, magnesium, rhodium, uranium, a transitionmetal, yttrium and the Lanthanide metals. The non-metallic ion cancomprise a ligand, chelate or complexing agent. For large metal ionssuch as the lanthanides(III), ligands that provide six or more donoratoms may be used. Ligands for any of the metal ions within the scope ofthe present disclosure need not form thermodynamically stable complexeswith the metal ions. It may be sufficient that they are kineticallyinert to metal ion release. Accordingly, macrocyclic ligands can formkinetically inert complexes with labile metal ions if properly designed.Also, tetraazamacrocycle ligands strongly chelate the transition metalions and Zn(II). The chemical degradation may be performed in thepresence of a divalent cation, for example, Mg²⁺ and Zn²⁺. The chemicaldegradation may be performed under a condition selected from the groupconsisting of a pH having value from 6 to 14, a pH having value from 0to 6, a temperature from 10° C. to 100° C., in the presence of a heavymetal ion, in the presence of a divalent cation, and any combinationthereof.

In yet another aspect, the present disclosure provides a method forprocessing a biological sample. The method may comprise providing thebiological sample comprising a RNA molecule in a 3D matrix. The RNAmolecule may comprise a nucleic acid sequence. Next, a primer may behybridized to the RNA molecule. The primer may be a reversetranscription primer. The primer may not include a functional moiety forimmobilization to the 3D matrix. Next, a reverse transcriptase may beused to reverse transcribe the RNA molecule by extending the primer togenerate a cDNA molecule hybridized to the RNA molecule in thebiological sample. The cDNA molecule may comprise a functional moietythat can immobilize the cDNA molecule to the 3D matrix.

In some cases, the RNA molecule hybridized to the cDNA molecule may bedegraded to provide the cDNA molecule immobilized to the 3D matrixthrough the functional moiety. The cDNA molecule may comprise anadditional nucleic acid sequence that is a reverse complement of thenucleic acid sequence.

The RNA molecule may comprise a functional moiety. The RNA molecule maybe immobilized to the 3D matrix via the functional moiety.

Various methods may be used to degrade the RNA molecule. For example,the RNA molecule may be degraded by a non-ribonuclease enzyme. Thenon-ribonuclease enzyme can be a reverse transcriptase or a DNA bindingprotein. For another example, the RNA molecule may be degraded by anon-enzymatic reaction. The non-enzymatic reaction may be performedunder a condition selected from the group consisting of a pH havingvalue from 6 to 14, a temperature from 10° C. to 100° C., in thepresence of a heavy metal ion, and any combination thereof.

The cDNA molecule may be contacted with a probe. The probe may comprisea region that is not hybridizable with said cDNA molecule. The regionthat is not hybridizable with said cDNA molecule may be a terminalregion or an internal region between two terminal regions.

The probe may be a padlock probe, wherein the padlock probe comprises 5′and 3′ terminal regions complementary to the cDNA molecule. The 5′ and3′ terminal regions of the padlock probe may be hybridized to the cDNAmolecule. The padlock probe may be circularized by coupling two ends ofthe padlock probe together to yield a circularized padlock probe. Forexample, the two ends of the padlock probe can be ligated together by aligase. A nucleic acid sequence of the circularized padlock probe or aderivative thereof may be detected, thereby identifying the nucleic acidsequence of the RNA molecule. For example, the nucleic acid sequence ofthe circularized padlock probe or a derivative thereof can be detectedby nucleic acid hybridization or sequencing.

Upon hybridizing the two ends of padlock probe and the cDNA molecule,the two ends of the padlock probe may be contiguous or separated by agap region of the cDNA molecule. The gap region may be of variouslengths. For example, the gap region can comprise at least 1, at least5, at least 10, at least 15, at least 20, at least 25, at least 30, atleast 35, at least 40, at least 45, or at least 50 nucleotide(s). Foranother example, the gap region can comprise from 2 to 10, from 10 to20, from 20 to 50, from 50 to 100, from 100 to 150, from 150 to 200,from 200 to 300, from 300 to 400, from 400 to 500 nucleotides. Acircularized padlock probe can be generated by ligating two ends of thepadlock probe together. In the cases where the two ends are contiguous,the two ends can be ligated directly. In the cases where the two endsare separated by a gap region, the gap region can be filled in beforeligation. The gap region can be filled in by incorporating one or morenucleotides in an extension reaction, for example, by extending from the3′ end of the two ends. The extension reaction can be performed by apolymerase. The gap region can also be filled in by hybridizing anadditional nucleotide or an additional oligonucleotide sequence to thegap region. The length of the additional oligonucleotide sequence can bedetermined based on the length of the gap region. For example, theadditional oligonucleotide sequence can be of the same length as the gapregion such that after hybridizing with the gap region, the 5′ end ofthe additional oligonucleotide sequence is adjacent to the 3′ end of thepadlock probe and the 3′ end of the additional oligonucleotide sequenceis adjacent to the 5′ end of the padlock probe. For example, theadditional oligonucleotide sequence can comprise from 2 to 10, from 10to 20, from 20 to 50, from 50 to 100, from 100 to 150, from 150 to 200,from 200 to 300, from 300 to 400, or from 400 to 500 nucleotides. Thecircularized padlock probe can then be generated by ligating the ends ofthe additional oligonucleotide sequence with the ends of the padlockprobe.

The circularized padlock probe may be further subjected to amplificationto generate an amplification product of a sequence of the circularizedpadlock probe. For example, the circularized padlock probe may besubjected to rolling circle amplification (RCA). The amplificationproduct may comprise a nucleic acid sequence corresponding to thenucleic acid sequence of the RNA molecule. The nucleic acid sequence ofthe amplification product may be detected, thereby identifying thenucleic acid sequence of the RNA molecule.

The probe may comprise a functional moiety. The probe may be immobilizedto the 3D matrix via the functional moiety. The functional moiety may bedirectly conjugated on the probe. The functional moiety may beintroduced to the probe during synthesis of the probe. For example, anucleotide analog having a functional moiety or a precursor of thefunctional moiety may be incorporated to a growing chain of the probeduring synthesis. The functional moiety may be introduced to the probethrough a tethering oligonucleotide. The probe may hybridize to thetethering oligonucleotide comprising the functional moiety. Thetethering oligonucleotide may hybridize to the region of the probe thatis not hybridizable to the cDNA molecule. A sequence of the probe or aderivative thereof may be detected, thereby identifying the nucleic acidsequence of the RNA molecule.

The cDNA molecule may comprise a functional moiety. The reversetranscriptase may be used to incorporate a nucleotide analog comprisingthe functional moiety into a growing strand, to yield the cDNA moleculecomprising the nucleotide analog. For example, the nucleotide analogcomprises amino-allyl dUTP, 5-TCO-PEG4-dUTP, C8-Alkyne-dUTP,5-Azidomethyl-dUTP, 5-Vinyl-dUTP, 5-Ethynyl dUTP, or a combinationthereof.

The primer or the cDNA molecule may be modified, after generating thecDNA molecule, to include the functional moiety. The primer may bemodified to include the functional moiety prior to generating the cDNAmolecule. For example, the primer may be modified with the functionalmoiety through conjugation chemistry. The primer may comprise a regionthat is not hybridizable to the RNA molecule. The region that is nothybridizable to the RNA molecule may be coupled to a tetheringoligonucleotide that comprises a functional moiety. For example, theregion may hybridize to the tethering oligonucleotide that comprises thefunctional moiety.

The functional moiety may be attached to the cDNA molecule through anenzymatic reaction or a non-enzymatic reaction. The enzymatic reactionmay comprise using an enzyme to attach a nucleotide or anoligonucleotide having the functional moiety to the cDNA molecule. Theenzyme can be a ligase, a polymerase, or a combination thereof. Thenon-enzymatic reaction may comprise attaching a chemical reagent havingthe functional moiety to the cDNA molecule by alkylation oroxymercuration. The cDNA molecule may be coupled to a tetheringoligonucleotide having a functional moiety. For example, the cDNAmolecule may hybridize to a tethering oligonucleotide having afunctional moiety. The tethering oligonucleotide may hybridize to theprimer.

The 3D matrix may further comprise an additional functional moiety. Theadditional functional moiety may react with the function moiety of thecDNA molecule, thereby immobilizing the cDNA molecule.

In a further aspect, the present disclosure features a kit comprisingsome or all of the reagents, enzymes, probes, and primers necessary toperform the methods described herein. The items comprising the kit maybe supplied in separate vials or may be mixed together, whereappropriate.

FIG. 1 shows an example of a method for identification of a nucleic acidsequence in a biological sample. In a first operation 101, thebiological sample comprising a ribonucleic acid (RNA) moleculehybridized to a deoxyribonucleic acid molecule (DNA) in athree-dimensional (3D) matrix may be provided. The RNA molecule maycomprise a nucleic acid sequence to be identified. Next, in a secondoperation 102, a reverse transcriptase may be used to degrade or digestat least a portion of the RNA molecule hybridized to the DNA molecule.The DNA molecule may comprise an additional nucleic acid sequence thatis a reverse complement of the nucleic acid sequence. Next, in a thirdoperation 103, the additional nucleic acid sequence in the biologicalsample may be detected, thereby identifying the nucleic acid sequence.

FIG. 2 shows an example of a method for identification of a nucleic acidsequence in a biological sample. In a first operation 201, thebiological sample comprising a ribonucleic acid (RNA) moleculehybridized to a deoxyribonucleic acid molecule (DNA) in athree-dimensional (3D) matrix may be provided. The RNA molecule maycomprise a nucleic acid sequence to be identified. Next, in a secondoperation 202, a deoxyribonucleic acid (DNA) binding protein that is nota reverse transcriptase or a ribonuclease may be used to degrade ordigest at least a portion of the RNA molecule hybridized to the DNAmolecule. The DNA molecule may comprise an additional nucleic acidsequence that is a reverse complement of the nucleic acid sequence.Next, in a third operation 203, the additional nucleic acid sequence inthe biological sample may be detected, thereby identifying the nucleicacid sequence.

FIG. 3 shows an example of a method for identification of a nucleic acidsequence in a biological sample. In a first operation 301, thebiological sample comprising a ribonucleic acid (RNA) moleculehybridized to a deoxyribonucleic acid molecule (DNA) in athree-dimensional (3D) matrix may be provided. The RNA molecule maycomprise a nucleic acid sequence to be identified. Next, in a secondoperation 302, at least a portion of the RNA molecule hybridized to theDNA molecule may be degraded non-enzymatically. The DNA molecule maycomprise an additional nucleic acid sequence that is a reversecomplement of the nucleic acid sequence. Next, in a third operation 303,the cDNA molecule may be contacted by a probe. Next, in a fourthoperation 304, a sequence of the probe or a derivative thereof may bedetected, thereby identifying the nucleic acid sequence of the RNAmolecule.

FIG. 4 shows an example of a method for processing a biological sample.In a first operation 401, the biological sample comprising a ribonucleicacid (RNA) molecule in a three-dimensional (3D) matrix may be provided.The RNA molecule may comprise a nucleic acid sequence. Next, in a secondoperation 402, a primer may be hybridized to the RNA molecule, whichprimer does not include a functional moiety for immobilization to thematrix. Next, in a third operation 403, a reverse transcriptase may beused to reverse transcribe the RNA molecule by extending the primer togenerate a complementary deoxyribonucleic acid (cDNA) moleculehybridized to the RNA molecule in the biological sample. The cDNAmolecule may comprise a functional moiety that immobilizes the cDNAmolecule to the 3D matrix.

In various embodiments, a target DNA or RNA molecule can hybridize to aprimer or a probe in the present of a hybridization reaction enhancingagent. The hybridization reaction enhancing agent can enhance a rate ofthe hybridization reaction between a target nucleic acid molecule and aprobe having sequence complementarity with the target sequence of thetarget molecule, as compared to another hybridization reaction conductedbetween the target nucleic acid molecule and the probe in the absence ofthe hybridization reaction enhancing agent. An example of ahybridization reaction enhancing agent may be dextran sulfate. However,enzymes may be strongly inhibited by dextran sulfate, and being a highlycharged, high molecular weight polymer, it may be difficult to washdextran sulfate from the sample to the point where the downstreamenzymatic reactions, e.g., reverse transcription, ligation, DNApolymerization, are not strongly inhibited. In the absence of ahybridization reaction enhancing agent, however, the kinetics of in situhybridization may be orders of magnitude slower. Therefore, ahybridization reaction enhancing agent that does not strongly inhibitenzymatic reactions can be used in the methods described herein. Thehybridization reaction enhancing agents may be high-molecular weight,high valency charged polymers. For example, hybridization reactionenhancing agent may be polymers such as polyacrylic acid,polyvinylsulfonic acid, and alginate. The hybridization reactionenhancing agents may be polymers similar to dextran sulfate.

In some instances, an intermolecular organization of the hybridizationreaction enhancing agents may be a factor in determining itseffectiveness as a hybridization reaction enhancing agent. As anexample, dextran sulfate can aid in the formation of networks (e.g.,highly localized concentrations of probes) during hybridization, thusexpediting the annealing process. The G-blocks of alginate canparticipate in intermolecular cross-linking with divalent cations (e.g.,Ca²⁺) to form hydrogels. Dextran sulfate may not form hydrogels otherthan under exogenous chemical cross-linking reactions and in thepresence of chitosan, neither of which may be present during typicalnucleic acid hybridization reactions. In some instances, thehybridization reaction enhancing agent may self-associate in theformation of hydrogels. Alternatively, the hybridization reactionenhancing agent may not self-associate in the formation of hydrogels.The difference in the ability to self-associate in the formation ofhydrogels may explain the difference between dextran sulfate andalginate in improving the kinetics of nucleic acid DNA hybridizationreactions.

For example, polyacrylic acid and polyvinylsulfonic acid may botheffectively function as a hybridization reaction enhancing agent whilealginate may not. This may be due to the intermolecular organization,which can reduce its effectiveness in crowding DNA. In some instances,polyacrylic acid may inhibit enzymatic reactions, but polyvinylsulfonicacid may exhibit much less inhibition. As an example, one mechanism ofinhibition may be via chelation of essential metal or charged cofactors,such as Mg²⁺, Ca²⁺, Mn²⁺, Na⁺, phosphate, and other metal and chargedions, which are required for enzyme function. A polyion salt such assodium polyacrylic acid may exchange sodium ions for magnesium ions inthe presence of a magnesium-containing enzyme reaction buffer, reducingthe effective concentration of the essential cofactor. Another mechanismof inhibition may be binding and structural damage to the enzyme, e.g.the charge attraction and binding between charged domains of the enzymeand the ionic polymer, which may cause effective sequestration of theenzyme within the reaction, as well as disrupt electrostatic or chargeinteractions within the enzyme, which may be required for enzymestructure and related function. Wettability, or hydrophobicity, charge,and structure may alternatively, or additionally contribute to thestrength of polyion-protein interactions. Protein absorption on thepolyion or within the polyionic network may contribute to effectivedecrease in enzyme concentration.

In some embodiments, the property or activity of the hybridizationreaction enhancing agent can be controlled (e.g., inactivated). Forexample, some polymers that function as the hybridization reactionenhancing agent can comprise charged groups and subsequently the chargedgroups can be cleaved off or neutralized. This process can convert thepolymers into neutral polymers such as PEG, which can enhance theefficiency of enzymatic reactions. In some embodiments, polymers thatfunction as the hybridization reaction enhancing agents can bespecifically degraded into small monomers and can be easily removed fromthe sample such as by washing away from the sample. The chemistry ofpassivation or degradation of the hybridization reaction enhancing agentmay need to be orthogonal to nucleic acids, i.e., not degrading nucleicacids or rendering nucleic acids incompatible. Some of these functionalgroups include alpha-hydroxy acids, which can be cleaved by sodiumperiodate; beta-keto acids, which can be cleaved with heat;phosphorothioate linkages, which can be cleaved with silver ions;disulfide linkages, which can be cleaved by reduction into thiols; andother types of chemical linkages which may be cleaved by photo- orchemical treatment.

Examples of polyions or polyelectrolytes for enzyme-compatibleenhancement of nucleic acid hybridization kinetics includepolycondensation reactions of Cys(Lys)nCys, polymers such as PEG, PVA,or PAA, which may be subsequently modified via a cleavable linker toinclude chemical groups conferring ionic charge, or polymers formed frommonomers including cleavable linkages, such that the polymer may bedegraded subsequent to functioning as a hybridization reaction enhancingagent. As an alternative to ionic charge, these polymers may includenon-ionic groups that become hydrated in solution, which can enhancenucleic acid hybridization rates by molecular crowding and/orsequestration of water.

In some cases, the hybridization reaction enhancing agent may not becontrolled. For example, the hybridization reaction enhancing agent maynot comprise a cleavable functional group such that it can beinactivated or degraded into monomers. In these cases, the hybridizationreaction enhancing agents may be removed from the sample afterfunctioning as the hybridization reaction enhancing agent to enhancenucleic acid hybridizations. For example, the hybridization reactionenhancing agent can be washed away from the sample or the 3D matrix. Thewash step may be performed after hybridizing a primer or a probe to atarget nucleic acid molecule and before any subsequent enzymaticreactions such as reverse transcription, ligation, and amplification.

The present disclosure provides methods to enhance the hybridization ofprobes for capturing RNA, cDNA, and DNA species onto the 3D matrix fordetection via FISSEQ. The hybridization reaction enhancing agent can beadded in the hybridization buffer. In some embodiments, a hybridizationbuffer containing high salt, such as SSC (sodium chloride sodium citratebuffer), can be used. In some embodiments, a hybridization buffercontaining blocking agents can be used. The blocking agents can reducenon-specific binding of probes to off-target sequences and/or bypreventing electrostatic interactions with other components of thesample, such as yeast tRNA, salmon sperm, detergents such as Triton-X,Tween 20, SPAN, peptides such as BSA, and other agents such as Ficoll.In some embodiments, a hybridization buffer containing agents whichalter the annealing properties of DNA, such as the melting temperature.An example of agents that can alter the annealing temperature includesformamide. The hybridization reaction enhancing agent can have anaverage molecular weight of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, or more kDa. Insome embodiments, a hybridization reaction enhancing agent can presentin at least about 1%, 5%, 10%, 15%, 20%, or more weight per volume inthe reaction.

The hybridization reaction enhancing agent can be a polyionic, apolyelectrolyte, a hydrophilic or a hydrating polymer. The hybridizationreaction enhancing agent can comprise a polymer backbone and one or morehydrating groups. The hydrating groups can be ionic, electrolytic, orhydrophilic. In some embodiments, the hydrating groups may bespecifically inactivated, e.g., as by rendering an ionic group to haveneutral charge, or as by rendering a strongly hydrating group to beweakly hydrating. The inactivation chemistry can be substantiallynonreactive with RNA, DNA, proteins, and/or other types of biomolecules.The inactivated polymer is compatible with enzymatic reactions.

The hybridization reaction enhancing agent can comprise a cleavablelinkage between the polymer backbone and the hydrating group. Thecleavable linkages can comprise alpha-hydroxy acids, which can becleaved by sodium periodate. The cleavable linkages comprise beta-ketoacids, which can be cleaved with heat. The cleavable linkages cancomprise phosphorothioate linkages, which can be cleaved with silverions. The cleavable linkages can comprise disulfide linkages, which canbe cleaved by reduction into thiols. Other types of chemical linkagesmay be cleaved by photo- or chemical treatment. In some cases, thehybridization reaction enhancing agent can comprise cleavable linkagesalong the backbone of the polymer, where the cleavable linkages can beany types of cleavable linkages described herein.

The methods provided herein can comprise the use of a hybridizationreaction enhancing agent in sample processing for targeted RNA or DNAdetection. In some cases, a plurality of probes can be hybridized insitu using a hybridization buffer containing one of the hybridizationreaction enhancing agents described herein.

The methods described herein may comprise hybridizing a plurality ofprobes in situ using a hybridization buffer containing one of thehybridization reaction enhancing agents described herein. In someembodiments, the methods further comprise triggering cleavage of thecleavable groups of the hybridization reaction enhancing agents toinactivate the hybridization reaction enhancing agent. In some otherembodiments, the methods further comprise removing the hybridizationreaction enhancing agents from the sample or the 3D matrix withoutinactivating the hybridization reaction enhancing agents.

Computer Systems

The present disclosure provides computer systems that are programmed toimplement methods of the disclosure. FIG. 5 shows a computer system 501that is programmed or otherwise configured to process a biologicalsample and/or identify a nucleic acid sequence in a biological sample.The computer system 501 can regulate various aspects of componentsand/or devices of the present disclosure utilized in detection ofnucleic acid sequences in a biological sample and/or sample processing,such as, for example, light sources, detectors (e.g., light detectors),devices or components utilized for releasing agents, devices orcomponents utilized in providing conditions for reactions (e.g.,hybridization, sequencing, enzymatic reactions), etc. The computersystem 501 can be an electronic device of a user or a computer systemthat is remotely located with respect to the electronic device. Theelectronic device can be a mobile electronic device.

The computer system 501 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 505, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 501 also includes memory or memorylocation 510 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 515 (e.g., hard disk), communicationinterface 520 (e.g., network adapter) for communicating with one or moreother systems, and peripheral devices 525, such as cache, other memory,data storage and/or electronic display adapters. The memory 510, storageunit 515, interface 520 and peripheral devices 525 are in communicationwith the CPU 505 through a communication bus (solid lines), such as amotherboard. The storage unit 1115 can be a data storage unit (or datarepository) for storing data. The computer system 501 can be operativelycoupled to a computer network (“network”) 530 with the aid of thecommunication interface 520. The network 530 can be the Internet, aninternet and/or extranet, or an intranet and/or extranet that is incommunication with the Internet. The network 530 in some cases is atelecommunication and/or data network. The network 530 can include oneor more computer servers, which can enable distributed computing, suchas cloud computing. The network 530, in some cases with the aid of thecomputer system 501, can implement a peer-to-peer network, which mayenable devices coupled to the computer system 501 to behave as a clientor a server.

The CPU 505 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 510. The instructionscan be directed to the CPU 505, which can subsequently program orotherwise configure the CPU 505 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 505 can includefetch, decode, execute, and writeback.

The CPU 505 can be part of a circuit, such as an integrated circuit. Oneor more other components of the system 501 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 515 can store files, such as drivers, libraries andsaved programs. The storage unit 515 can store user data, e.g., userpreferences and user programs. The computer system 501 in some cases caninclude one or more additional data storage units that are external tothe computer system 501, such as located on a remote server that is incommunication with the computer system 501 through an intranet or theInternet.

The computer system 501 can communicate with one or more remote computersystems through the network 530. For instance, the computer system 501can communicate with a remote computer system of a user (e.g., a userperforming sample processing or nucleic acid sequence detection of thepresent disclosure). Examples of remote computer systems includepersonal computers (e.g., portable PC), slate or tablet PC's (e.g.,Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g.,Apple® iPhone, Android-enabled device, Blackberry®), or personal digitalassistants. The user can access the computer system 501 via the network530.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 501, such as, for example, on the memory510 or electronic storage unit 515. The machine executable or machinereadable code can be provided in the form of software. During use, thecode can be executed by the processor 505. In some cases, the code canbe retrieved from the storage unit 515 and stored on the memory 510 forready access by the processor 505. In some situations, the electronicstorage unit 515 can be precluded, and machine-executable instructionsare stored on memory 510.

The code can be pre-compiled and configured for use with a machinehaving a processor adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 501, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 501 can include or be in communication with anelectronic display 535 that comprises a user interface (UI) 540 forproviding, for example, protocols to perform the sample processingmethods and/or nucleic acid sequence detection methods described in thepresent disclosure. Examples of UI's include, without limitation, agraphical user interface (GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 505. Thealgorithm can, for example, be executed so as to detect a nucleic acidsequence utilizing methods and systems disclosed in the presentdisclosure. Optionally, the algorithms may be executed so as to controlor effect operation of a component (e.g., light source, detector,reagent flow, etc) of the systems described herein to effect detectionof a nucleic acid sequence.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

EXAMPLE Example I Immobilizing, Amplifying and Imaging DNA/RNA Moleculeswithin Cells

Human iPS cells or human primary fibroblasts will be grown on a 1.5cover slip. They will be fixed using 4% formaldehyde in PBS for 15 min,followed by three washes of 70% ethanol. The reverse transcriptionmixture containing 1 μM random hexamer or 0.1 μM polydT(18)V primer withadditional adapter sequences (TCTCGGGAACGCTGAAGA), 250 μM dNTP, 40 μMaminoallyl dUTP (Anaspec), 20 U RNase inhibitor and 100 U MMuLV reversetranscriptase (Enzymatics) will then be added to the fixed cells andincubated overnight at 37° C. The sample will then be washed using PBS,and cross-linked using 100 μM BS(PEG)9 (Thermo-Fisher Scientific) in PBSfor 1 hour, followed by 1M Tris treatment for 15 min. Thecircularization mixture containing 25 U CircLigase (Epicentre), 1 mMMnCl and 1 M Betaine will be added, and the sample will be incubated at60° C. for 2 hours. The RNA will be degraded using Avian myeloblastosisvirus (AMV) reverse transcriptase at 37° C. for 1 hour. Alternatively,the RNA will be degraded using a solution having a pH from 8 to 10, orby heating the sample at 100° C. for about 20 min. The RCA primer isthen hybridized to the sample at 60° C. for 15 min and washed. Forrolling circle amplification, 100 U phi29 DNA polymerase (Enzymatics),250 μM dNTP and 40 μM aminoallyl dNTP will be added to the sample andincubated at 30° C. overnight. The sample will then be washed using PBS,and cross-linked using 100 μM BS(PEG)9 in PBS for 1 hour, followed by 1MTris treatment for 15 min. For the DNA amplicon detection, 1 μMfluorescently label oligonucleotides will be diluted in 2×SSC andhybridized to the matrix containing the DNA amplicons at 60° C. andwashed. Imaging can be done using Leica SP5 scanning confocal microscopeusing 10×, 20× or 63× objectives in four color channels (FITC, Cy3,Texas Red and Cy5). The image stacks containing up to 50 opticalsections can then be visualized using Imaris Bitplane software forthree-dimensional reconstruction of the DNA amplicons within the samplematrix.

Methods described herein allow one to immobilize, amplify and imagesingle DNA/RNA molecules in a three-dimensional space without perturbingthe structure. DNA/RNA can be amplified in situ. The DNA/RNA can beco-polymerized into a matrix material in situ, and individual ampliconscan be interrogated/hybridized with fluorescent oligonucleotides andimaged. When viewed under much higher magnification, individualamplicons can be imaged using confocal microscopy. This allows one tofind out where different DNA/RNA molecules reside, how they arecompartmentalized among different cell types and morphologies and howtheir representation changes over time in developing tissues. Thesimilar concept can be used for many other specimens in both natural andsynthetic materials, as long as they can be co-polymerized and/orencapsulated by the DNA amplicons.

According to one specific aspect, inside individual mammalian cells, 20to 500K mRNA molecules may be distributed throughout the cytoplasm.Cells can be fixed and permeabilized. Cellular RNA can then be convertedinto cDNA molecules using dUTP in place or in addition to dTTP. The cDNAmolecules containing modified dUMP residues can then be cross-linked toeach other and circularized, forming a three-dimensional pseudo-polymerof circular cDNA molecules inside individual cells. Then rolling circleamplification can be used to amplify the cDNA network into a DNAamplicon network. This cell-based DNA amplicon network then storesinformation about each transcript's identity, location,variation/mutations, etc. The cell-based DNA amplicon matrix can be readusing sequencing by ligation (i.e. ABI SoLiD), sequencing by synthesis(i.e. Illumina), or any other proprietary or open sequencingchemistries. Sequencing may be whole genome sequencing or targetedsequencing. Sequencing may be performed by massively parallel arraysequencing (e.g., Illumina) or single molecule sequencing (e.g., PacificBiosciences of California or Oxford Nanopore). Given thethree-dimensional nature of the DNA amplicon network, one can useconfocal or multi-photon microscopy to sequencing individual ampliconsthroughout the whole thickness of the amplicon network, enabling one tovisualize the cDNA distribution of transcripts between the apical sideand the basal side of the cells. Given the tight packing density, onecan selectively read different subpopulations sequentially, reducing thedensity of information read at any given time and extending over timefor better spatial resolution.

Example II Sample Processing and Target Detection within a 3D Matrix

Mouse brain tissue samples were sectioned onto a glass slide. They werefixed using 4% formaldehyde in PBS for 20 min, followed by quenching in100 mM glycine for 15 min and a 5 min 1×PBS rinse. Samples were thengradually dehydrated in ethanol and incubated overnight in 100% ethanol.Samples were gradually rehydrated to 1×PBST, and permeabilized in 0.2%TritonX-100. 1 μM tetherable reverse transcription primers werehybridized overnight at 37° C. in a buffer containing 2×SSC and 10%dextran sulfate. Samples were washed for 20 min at 37° C. to removeexcess oligonucleotides, embedded in a polyacrylamide matrix, andproteins cleared overnight at 37° C. with 16 U/mL of proteinase K in 4%SDS buffer, pH 7.3. To remove residual hybridization reaction enhancingagents and proteinase, samples were washed for several hours in 1×PBST,replacing buffer every 30 min. The reverse transcription mixturecontaining 1.25 mM dNTP, 1 U/μL RNase Inhibitor (Enzymatics), and 10U/μL EnzScript reverse transcriptase (Enzymatics) were added to clearedsections and incubated overnight at 37° C. ssRNA and RNA duplexed withcDNA product were removed via borate buffer-mediated chemical hydrolysisperformed at 55° C. for 2 hours, and then DNA padlock oligonucleotideprobes were hybridized to ss-cDNA during an overnight incubation at 37°C. in a buffer containing 2×SSC and 10% dextran sulfate. Samples werewashed for 20 min 37° C. to remove unbound DNA padlock oligonucleotideprobes, then further washed for several hours in 1×PBST, replacingbuffer every 30 min, to remove residual hybridization reaction enhancingagents. DNA padlock oligonucleotide probes were ligated by 30 U/μL of T7ligase (Enzymatics) at room temperature for 60 min, then rolling circleamplification was performed overnight at 30° C. in the presence of 0.5U/μL phi29 DNA polymerase (Enzymatics), 625 μM dNTPs, and 0.025 μMcompaction oligonucleotides. For the DNA amplicon detection, 1 μMfluorescently label oligonucleotides were diluted in 2×SSC andhybridized to the matrix containing the DNA amplicons at 60° C. andwashed. Imaging was done using ReadCoor automated fluorescent in situsequencing device. The 3D image data were processed to identify eachamplicon and visualized using a web-browser based software tool forthree-dimensional reconstruction of the DNA amplicons within the matrix.FIG. 6 shows an example image of the mouse brain tissue sample 600processed and imaged using the method described above. Cell nuclei(e.g., 601 of FIG. 6) are shown as bigger bright spots across the image.Sequencing readout of fluorescent signals of amplicons (e.g., 602 ofFIG. 6) are shown as smaller and dimmer spots across the image. Shown inthe image is the sequencing readout of one sequencing cycle. The samplewas subjected to multiple sequencing cycles to determine the sequencesof bases for each amplicon.

What is claimed is:
 1. A method for identification of a nucleic acidsequence in a biological sample, comprising: (a) providing saidbiological sample comprising a ribonucleic acid (RNA) moleculehybridized to a deoxyribonucleic acid (DNA) molecule in athree-dimensional (3D) matrix, wherein said RNA molecule comprises saidnucleic acid sequence, and wherein said DNA molecule comprises anadditional nucleic acid sequence that is a reverse complement of saidnucleic acid sequence; (b) degrading or digesting at least a portion ofsaid RNA molecule hybridized to said DNA molecule, wherein said at leastsaid portion of said RNA molecule is degraded or digestednon-enzymatically at conditions suitable for conducting a clickreaction; and (c) detecting said additional nucleic acid sequence,thereby identifying said nucleic acid sequence.
 2. The method of claim1, wherein said DNA molecule is immobilized to said 3D matrix.
 3. Themethod of claim 2, wherein said DNA molecule comprises a functionalmoiety, and wherein said DNA molecule is immobilized to said 3D matrixvia said functional moiety.
 4. The method of claim 1, wherein said RNAmolecule is immobilized to said 3D matrix.
 5. The method of claim 4,wherein said RNA molecule comprises a functional moiety, and whereinsaid RNA molecule is immobilized to said 3D matrix via said functionalmoiety.
 6. The method of claim 1, wherein (c) comprises contacting saidDNA molecule with a probe.
 7. The method of claim 6, wherein said probecomprises a functional moiety, and wherein said probe is immobilized tosaid 3D matrix via said functional moiety.
 8. The method of claim 6,wherein said probe is a padlock probe, wherein said padlock probecomprises 5′ and 3′ terminal regions complementary to said DNA molecule,and wherein said 5′ and 3′ terminal regions of said padlock probe arehybridized to said DNA molecule.
 9. The method of claim 8, furthercomprising circularizing said padlock probe by ligating two ends of saidpadlock probe together, to yield a circularized padlock probe.
 10. Themethod of claim 9, wherein said two ends of said padlock probe arecontiguous.
 11. The method of claim 9, wherein said two ends of saidpadlock probe are separated by a gap region comprising at least onenucleotide.
 12. The method of claim 11, wherein said gap regioncomprises from 2 to 500 nucleotides.
 13. The method of claim 11, furthercomprising filling said gap region by incorporating at least onenucleotide in an extension reaction.
 14. The method of claim 11, furthercomprising filling said gap region by at least one additional nucleotideor an oligonucleotide sequence.
 15. The method of claim 9, furthercomprising subjecting said circularized padlock probe to rolling circleamplification (RCA) to generate a nucleic acid molecule using saidcircularized padlock probe as a template, which nucleic acid moleculecomprises a sequence corresponding to said nucleic acid sequence of saidRNA molecule.
 16. The method of claim 15, further comprising detectingsaid sequence of said nucleic acid molecule, thereby identifying saidnucleic acid sequence of said RNA molecule.
 17. The method of claim 1,further comprising, prior to (a), reverse transcribing said RNA moleculeto generate said DNA molecule hybridized to said RNA molecule in saidbiological sample.
 18. The method of claim 17, wherein said RNA moleculeis reverse transcribed using a reverse transcriptase.
 19. The method ofclaim 17, further comprising, prior to (a), hybridizing a reversetranscription primer to said RNA molecule, wherein said reversetranscription primer comprises a functional moiety, and wherein said DNAmolecule is immobilized to said 3D matrix via said functional moiety.20. The method of claim 17, wherein (b) is performed under a first setof conditions and reverse transcribing said RNA molecule is performedunder a second set of conditions, wherein said first set of conditionsis different than said second set of conditions.
 21. The method of claim20, wherein said first set of conditions or said second set ofconditions is selected from the group consisting of pH, temperature,cofactor concentration, and cation concentration.
 22. The method ofclaim 1, wherein in (b) said at least said portion of said RNA moleculeis degraded or digested non-enzymatically in a presence of a metal ionsuitable for conducting said click reaction.
 23. The method of claim 22,wherein said metal ion is a copper ion.
 24. The method of claim 1,wherein said biological sample comprises a plurality of RNA molecules,wherein in (a) said plurality of RNA molecules has a fixed relative 3Dspatial relationship.
 25. The method of claim 1, wherein (b) comprisesdegrading said at least said portion of said RNA moleculenon-enzymatically by subjecting said at least said portion of said RNAmolecule to (1) a pH in a range from 6 to 14, (2) a temperature from 10°C. to 100° C., (3) a heavy metal ion, or (4) a divalent cation.
 26. Amethod for processing a biological sample, comprising: (a) providingsaid biological sample comprising a ribonucleic acid (RNA) molecule in athree-dimensional (3D) matrix, wherein said RNA molecule comprises anucleic acid sequence; (b) hybridizing a primer to said RNA molecule,which primer does not include a functional moiety for immobilization tosaid 3D matrix; (c) using a reverse transcriptase to reverse transcribesaid RNA molecule by extending said primer to generate a complementarydeoxyribonucleic acid (cDNA) molecule hybridized to said RNA molecule,which cDNA molecule comprises a functional moiety that immobilizes saidcDNA molecule to said 3D matrix; (d) contacting said cDNA molecule witha probe, which probe comprises an additional functional moiety; and (e)covalently or ionically coupling said additional functional moiety tosaid 3D matrix.
 27. The method of claim 26, further comprising degradingsaid RNA molecule hybridized to said cDNA molecule, to provide said cDNAmolecule immobilized to said 3D matrix through said functional moiety.28. The method of claim 26, wherein said probe comprises a sequenceregion that is not hybridizable with said cDNA molecule.
 29. The methodof claim 28, wherein said probe hybridizes to a tetheringoligonucleotide comprising said additional functional moiety.